Method of Storing a Separation Matrix

ABSTRACT

The present invention concerns a method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) providing a storage liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; b) permeating the separation matrix with the storage liquid; and c) storing the storage liquid-permeated separation matrix for a storage time of at least days. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

TECHNICAL FIELD

The present invention relates to a method of storing separation matricesthat comprise multimers of immunoglobulin-binding alkali-stabilizedProtein A domains covalently coupled to a porous support. The presentinvention further relates to separation matrix products and the use ofstorage liquids comprising at least 50% by volume of an aqueous alkalimetal hydroxide solution for the storage of separation matrices.

BACKGROUND ART

Immunoglobulins represent the most prevalent biopharmaceutical productsin either manufacture or development worldwide. The high commercialdemand for and hence value of this particular therapeutic market has ledto the emphasis being placed on pharmaceutical companies to maximize theproductivity of their respective immunoglobulin manufacturing processeswhilst controlling the associated costs.

Affinity chromatography is used in most cases, as one of the key stepsin the purification of these immunoglobulin molecules, such asmonoclonal antibodies (mAbs) or polyclonal antibodies (pAbs). Aparticularly interesting class of affinity reagents is proteins capableof specific binding to invariable parts of an immunoglobulin molecule,such interaction being independent on the antigen-binding specificity ofthe antibody. Such reagents can be widely used for affinitychromatography recovery of immunoglobulins from different samples suchas but not limited to serum or plasma preparations or cell culturederived feed stocks. An example of such a protein is staphylococcalprotein A, containing domains capable of binding to the Fc and Fabportions of IgG immunoglobulins from different species. These domainsare commonly denoted as the E-, D-, A-, B- and C-domains.

Staphylococcal protein A (SpA) based reagents have due to their highaffinity and selectivity found a widespread use in the field ofbiotechnology, e.g. in affinity chromatography for capture andpurification of antibodies as well as for detection or quantification.At present, SpA-based affinity medium probably is the most widely usedaffinity medium for isolation of monoclonal antibodies and theirfragments from different samples including industrial cell culturesupernatants. Accordingly, various matrices comprising protein A-ligandsare commercially available, for example, in the form of native protein A(e.g. Protein A SEPHAROSE™, GE Healthcare, Uppsala, Sweden) and alsocomprised of recombinant protein A (e.g. rProtein A SEPHAROSE™, GEHealthcare). More specifically, the genetic manipulation performed inthe commercial recombinant protein A product is aimed at facilitatingthe attachment thereof to a support and at increasing the productivityof the ligand.

An ongoing trend in the biopharmaceutical industry is the use ofversatile multi-product production facilities instead of single-productproduction facilities, allowing production-on-demand ofbiopharmaceuticals and a greater product variety, e.g. personalized ororphan biopharmaceuticals. Production campaigns in such multi-productfacilities are shorter and there is a need to effectively store theaffinity separation matrix between campaigns.

A common medium for storing separation matrices between campaigns issodium hydroxide. According to the PDA Biotechology Cleaning ValidationCommittee, concentrations of 0.1 to 1.0 M sodium hydroxide are commonfor storing packed chromatography columns. However, such storageconditions are associated with exposing the matrix to solutions withpH-values above 13 for long periods. For many affinity chromatographymatrices containing proteinaceous affinity ligands such alkalineenvironment is a very harsh condition and consequently results indecreased capacity of the affinity separation matrix owing toinstability of the ligand to the high pH involved.

An extensive research has therefore been focused on the development ofengineered protein ligands that exhibit an improved capacity towithstand alkaline pH-values. For example, Gulich et al. (SusanneGulich, Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober,Journal of Biotechnology 80 (2000), 169-178) suggested proteinengineering to improve the stability properties of a Streptococcalalbumin-binding domain (ABD) in alkaline environments. Gulich et al.created a mutant of ABD, wherein all the four asparagine residues havebeen replaced by leucine (one residue), aspartate (two residues) andlysine (one residue). Further, Gulich et al. report that their mutantexhibits a target protein binding behavior similar to that of the nativeprotein, and that affinity columns containing the engineered ligand showhigher binding capacities after repeated exposure to alkaline conditionsthan columns prepared using the parental non-engineered ligand. Thus, itis concluded therein that all four asparagine residues can be replacedwithout any significant effect on structure and function.

Recent work shows that changes can also be made to protein A (SpA) toeffect similar properties. US patent application publication US2005/0143566, which is hereby incorporated by reference in its entirety,discloses that when at least one asparagine residue is mutated to anamino acid other than glutamine or aspartic acid, the mutation confersan increased chemical stability at pH-values of up to about 13-14compared to the parental SpA, such as the B-domain of SpA, or Protein Z,a synthetic construct derived from the B-domain of SpA (U.S. Pat. No.5,143,844, incorporated by reference in its entirety). The authors showthat when these mutated proteins are used as affinity ligands, theseparation media as expected can better withstand cleaning proceduresusing alkaline agents. Further mutations of protein A domains with thepurpose of increasing the alkali stability have also been published inU.S. Pat. No. 8,329,860, JP 2006304633A, U.S. Pat. No. 8,674,073, US2010/0221844, US 2012/0208234, U.S. Pat. No. 9,051,375, US 2014/0031522,US 2013/0274451 and WO 2014/146350, all of which are hereby incorporatedby reference in their entireties. However, the currently availablemutants are still sensitive to alkaline pH and the correspondingaffinity separation matrices are therefore typically stored in 20%ethanol solution or 2% benzyl alcohol solution.

There is thus still a need in this field to obtain a separation matrixcontaining protein ligands having a further improved stability towardsalkaline storage procedures. There is also a need for such separationmatrices with an improved binding capacity to allow for economicallyefficient purification of therapeutic antibodies.

SUMMARY OF THE INVENTION

The inventors of the present invention have recognised that alcoholsolutions are suboptimal for the storage of affinity separationmatrices. Alcohols are flammable, subject to regulation, and difficultto dispose of. The inventors have recognised that aqueous alkali metalhydroxide solutions have a number of advantages as storage solutions.Alkali metal hydroxide solutions are bactericidal or bacteriostaticdepending on concentration. They can inactive most viruses, bacteria,yeasts, fungi and endotoxins. They are relatively cheap, easily disposedand removal from the separation matrix is simple to detect using pHand/or conductivity measurements.

It is therefore an object of the present invention to provide anaffinity separation matrix for the purification of immunoglobulins thatis stored in alkali metal hydroxide solution. It is a further object ofthe present invention to provide a method for storing an affinityseparation matrix for the purification of immunoglobulins in an alkalimetal hydroxide solution.

These objects are achieved by the method according to the appendedclaims of storing a separation matrix comprising multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains covalentlycoupled to a porous support. The alkali-stabilized Protein A domainscomprise mutants of a parental Fc-binding domain of StaphylococcusProtein A (SpA), as defined by, or having at least 80% such as at least90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO 52, wherein theamino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 areasparagines and wherein at least the asparagine residue at position 3 ofSEQ ID NO 51 or 52 has been mutated to an amino acid selected from thegroup consisting of glutamic acid, lysine, tyrosine, threonine,phenylalanine, leucine, isoleucine, tryptophan, methionine, valine,alanine, histidine and arginine. The method comprises the steps of:

-   -   a) providing a storage liquid comprising at least 50% by volume        of an aqueous alkali metal hydroxide solution;    -   b) permeating the separation matrix with the storage liquid; and    -   c) storing the storage liquid-permeated separation matrix for a        storage time of at least 5 days.

By using a separation matrix comprising multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains as definedabove, a highly alkali-stable separation matrix having a high dynamicbinding capacity is obtained. The inventors of the present inventionhave observed that such separation matrices are stable upon immersion inaqueous alkali metal hydroxide solution for extended periods such asfive days or more, and substantially retain dynamic binding capacityafter such prolonged immersion. This means that such a separationmatrices are suitable for storage in alkali metal hydroxide solutions.

Further mutations to the immunoglobulin-binding alkali-stabilizedProtein A domains may provide further enhancement of properties such asenhanced alkali stability. For example, the glutamine residue atposition 1 of SEQ ID NO 51 or 52 may be mutated to an alanine; and/orthe asparagine or glutamic acid residue at position 35 of SEQ ID NO 51or 52 may be mutated to an alanine.

The multimers of immunoglobulin-binding alkali-stabilized Protein Adomains may be homomultimers selected from the group consisting ofdimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers ornonamers. By using an appropriate multimer, the immunoglobulin bindingcapacity and alkali stability of the separation matrix may be increased.

The multimers of immunoglobulin-binding alkali-stabilized Protein Adomains may each comprise a C-terminal cysteine residue for covalentcoupling to the porous support. The multimers of immunoglobulin-bindingalkali-stabilized Protein A domains may be coupled to the porous supportvia thioether links. This provides a robust, alkali-stable andwell-proven method of attaching the ligands to the solid support.

The separation matrix may comprise at least 11 mg/ml, such as at least15 mg/ml, of the multimers of immunoglobulin-binding alkali-stabilizedProtein A domains covalently coupled to the porous support. This ensuresa separation matrix with a good binding capacity.

The porous support may comprise cross-linked polymer particles having avolume-weighted median diameter (d50,v) of 56-70 micrometers and a drysolids weight of 55-80 mg/ml. The porous support may for example behighly cross-linked agarose beads.

The aqueous alkali metal hydroxide solution used in the storage liquidmay be sodium hydroxide solution, potassium hydroxide solution or amixture thereof, preferably sodium hydroxide solution. Sodium hydroxidesolution is relatively cheap, readily available and widely accepted foruse as a storage solution. The aqueous alkali metal hydroxide solutionmay have a molarity of from 10 mM to 100 mM, such as from 30 mM to 50mM. This ensures a solution with a stable pH and good bacteriostatic orbactericidal properties.

The storage liquid may in some instances further comprise a C₂-C₇alcohol, such as ethanol, isopropanol or benzyl alcohol. A storageliquid combining an alcohol and an alkali metal hydroxide may be moreeffective in inactivating certain microorganisms, such as somespore-forming bacteria.

The storage liquid may comprise at least 70% by volume aqueous alkalimetal hydroxide solution, such as at least 90% by volume aqueous alkalimetal hydroxide solution, preferably at least 99% by volume aqueousalkali metal hydroxide solution.

In some instances the storage liquid may consist of, or consistessentially of, aqueous alkali metal hydroxide solution.

The minimum storage time for the separation matrix may be as short atime as storage is required, such as at least 5 days, such as at least10 days, such as at least 50 days, such as at least 100 days, or such asat least 200 days. The maximum storage time for the separation matrixmay be as long a time as storage is required, such as up to 400 days, orsuch as up to 700 days.

Prior to storing, the separation matrix may be cleaned and/or sanitizedwith a cleaning fluid, wherein the cleaning fluid comprises at least 50%by volume of an aqueous alkali metal hydroxide solution and wherein theaqueous alkali metal hydroxide solution has a molarity of from 500 mM to5 M, such as from 1 M to 2 M. The cleaning fluid may consist of, orconsist essentially of, aqueous alkali metal hydroxide solution. Thus,the separation matrix may be cleaned, sanitized and stored with littleor no requirement for using alcohols.

The separation matrix retains at least 80% of its original dynamicbinding capacity, such as at least 90% of its original dynamic bindingcapacity, after step b), i.e. after prolonged storage. Thus, theseparation matrix may be stored in aqueous alkali metal hydroxidesolution without subsequent excessive negative impact on its ability topurify immunoglobulins.

According to a further aspect of the present invention, the objects ofthe present invention are achieved by use of a storage liquid as definedin the appended claims. That is to say, use of a storage liquidcomprising at least 50% by volume of an aqueous alkali metal hydroxidesolution for the storage of a separation matrix comprising multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains covalentlycoupled to a porous support.

The storage liquid may be the same as the storage liquid previouslydescribed above in relation to the method of storing a separationmatrix. For example, it may comprise, consist essentially of, or consistof, sodium hydroxide solution having a molarity of from 10 mM to 100 mM,such as from 30 mM to 50 mM.

According to another aspect of the present invention, the objects of thepresent invention are achieved by a separation matrix product accordingto the appended claims. The separation matrix product comprises astorage receptacle, a separation matrix and a storage liquid. Thestorage receptacle contains the separation matrix permeated with thestorage liquid. The separation matrix comprises multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains covalentlycoupled to a porous support, wherein the alkali-stabilized Protein Adomains comprise mutants of a parental Fc-binding domain ofStaphylococcus Protein A (SpA), as defined by, or having at least 80%such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO51 or 52 are asparagines and wherein at least the asparagine residue atposition 3 of SEQ ID NO 51 or 52 has been mutated to an amino acidselected from the group consisting of glutamic acid, lysine, tyrosine,threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine,valine, alanine, histidine and arginine. The storage liquid comprises atleast 50% by volume of an aqueous alkali metal hydroxide solution.

Thus it is possible to package, store and transport separation matricesstored in aqueous alkali metal hydroxide solution. This avoids therequirement of storing in alcohol solution and thus avoids the need forusing volatile and flammable components in the storage liquid.

The storage receptacle may for example be a bottle, can or drum madefrom a liquid-impervious material such as plastic or glass. The storagereceptacle may also be a pre-packable column, i.e. a separation columnthat is filled with separation matrix at the production site.

The storage liquid may be the same as the storage liquid previouslydescribed above in relation to the method of storing a separationmatrix. For example, it may comprise, consist essentially of, or consistof, sodium hydroxide solution having a molarity of from 10 mM to 100 mM,such as from 30 mM to 50 mM.

Further objects, advantages and novel features of the present inventionwill become apparent to one skilled in the art from the followingdetailed description.

Definitions

The terms “antibody” and “immunoglobulin” are used interchangeablyherein, and are understood to include also fragments of antibodies,fusion proteins comprising antibodies or antibody fragments andconjugates comprising antibodies or antibody fragments.

The terms an “Fc-binding polypeptide”, “Fc-binding domain” and“Fc-binding protein” mean a polypeptide, domain or protein respectively,capable of binding to the crystallisable part (Fc) of an antibody andincludes e.g. Protein A and Protein G, or any fragment or fusion proteinthereof that has maintained said binding property.

The term “linker” herein means an element linking two polypeptide units,monomers or domains to each other in a multimer.

The term “spacer” herein means an element connecting a polypeptide or apolypeptide multimer to a support.

The term “% identity” with respect to comparisons of amino acidsequences is determined by standard alignment algorithms such as, forexample, Basic Local Alignment Tool (BLAST™) described in Altshul et al.(1990) J. Mol. Biol., 215: 403-410. A web-based software for this isfreely available from the US National Library of Medicine athttpliblast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome.Here, the algorithm “blastp (protein-protein BLAST)” is used foralignment of a query sequence with a subject sequence and determiningi.a. the % identity.

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like;“consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments.

BRIEF DESCRIPTION OF THE FIGURES

For a fuller understanding of the present invention and further objectsand advantages of it, the detailed description set out below should beread together with the accompanying figures, and in which:

FIG. 1 shows an alignment of the Fc-binding domains as defined by SEQ IDNO:1-7 and 51-52.

FIG. 2 shows results from Example 2 for the alkali stability of parentaland mutated tetrameric Zvar (SEQ ID NO 7) polypeptide variants coupledto an SPR biosensor chip.

FIG. 3 shows results from Example 4 for the alkali stability (0.5 MNaOH) of parental and mutated tetrameric Zvar (SEQ ID NO 7) polypeptidevariants coupled to agarose beads.

FIG. 4 shows results from Example 4 for the alkali stability (1.0 MNaOH) of parental and mutated tetrameric Zvar (SEQ ID NO 7) polypeptidevariants coupled to agarose beads.

FIG. 5 shows results from Example 7 for the alkali stability (1.0 MNaOH) of agarose beads with different amounts of mutated multimervariants (SEQ ID NO. 20) coupled. The results are plotted as therelative remaining dynamic capacity (Qb10%, 6 min residence time) vs.incubation time in 1 M NaOH.

FIG. 6 shows results from Example 7 for the alkali stability (1.0 MNaOH) of agarose beads with different amounts of mutated multimervariants (SEQ ID NO. 20) coupled. The results are plotted as therelative remaining dynamic capacity (Qb10%, 6 min residence time) after31 h incubation in 1 M NaOH vs. the ligand content of the prototypes.

FIG. 7 shows results from a pH gradient elution of polyclonal human IgGa) from the reference matrix MabSelect SuRe LX and b) a matrix accordingto the invention.

FIG. 8 shows analyses of the IgG1, IgG2 and IgG4 components in fractionsfrom the chromatograms of FIG. 7. a) reference matrix and b) matrixaccording to the invention. For each fraction, the first bar (blue)represents IgG1, the second (red) IgG 4 and the third (green) IgG 2.

FIG. 9 shows results from accelerated alkali stability measurements with1 M NaOH incubation for the reference matrix MabSelect SuRe LX (MSS LX)and a matrix according to the invention. The stability is expressed asthe percentage of the 10% breakthrough capacity remaining afterincubation.

FIG. 10 shows results from extended incubation with NaOH solutionshaving concentrations up to 50 mM for the reference matrix MabSelectSuRe (MSS) and a separation matrix according to the invention (Inv. Ex).The dynamic binding capacity at 10% breakthrough is shown for thematrices prior to and after alkali incubation.

DETAILED DESCRIPTION

One aspect of the present invention concerns a method of storing aseparation matrix comprising multimers of immunoglobulin-bindingalkali-stabilized Protein A domains covalently coupled to a poroussupport.

Throughout this detailed description, two separate numbering conventionsmay be used. Unless otherwise stated, the amino acid residue positionnumbering convention of FIG. 1 is used, and the position numbers aredesignated as corresponding to those in SEQ ID NO 4-7. This applies alsoto multimers, where the position numbers designate the positions in thepolypeptide units or monomers according to the convention of FIG. 1,unless otherwise stated. However, throughout the claims, summary ofinvention and on occasion in the detailed description, the positionnumbers corresponding to those of SEQ ID NO 51 and 52 are used. Notethat position 1 of SEQ ID NO 51 or SEQ ID NO 52 corresponds to position9 of SEQ ID NO 4-7, and in this manner the different numberingconventions may be interconverted.

The immunoglobulin-binding alkali-stabilized Protein A domains of theinvention, also termed herein as “the polypeptide”, comprise, consistessentially of, or consist of mutants of a parental Fc-binding domain ofStaphylococcus Protein A (SpA), as defined by, or having at least 80%such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO51 or 52 are asparagines and wherein at least the asparagine residue atposition 3 of SEQ ID NO 51 or 52 has been mutated to an amino acidselected from the group consisting of glutamic acid, lysine, tyrosine,threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine,valine, alanine, histidine and arginine.

SEQ ID NO 51 (truncated Zvar)QQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQ SEQ ID NO 52(truncated C domain) QQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKEILAEAKKLNDAQ

Such immunoglobulin-binding alkali-stabilized Protein A domains maycomprise, consist essentially of, or consist of mutants of a parentalFc-binding domain of Staphylococcus Protein A (SpA), as defined by, orhaving at least 90%, at least 95% or at least 98% identity to, SEQ IDNO: 1 (E-domain), SEQ ID NO: 2 (D-domain), SEQ ID NO:3 (A-domain), SEQID NO:22 (variant A-domain), SEQ ID NO: 4 (B-domain), SEQ ID NO: 5(C-domain), SEQ ID NO:6 (Protein Z), SEQ ID NO:7 (Zvar), SEQ ID NO 51(Zvar without the linker region amino acids 1-8 and 56-58) or SEQ ID NO52 (C-domain without the linker region amino acids 1-8 and 56-58) asillustrated in FIG. 1, wherein at least the asparagine (or serine, inthe case of SEQ ID NO 2) residue at the position corresponding toposition 11 in SEQ ID NO:4-7 has been mutated to an amino acid selectedfrom the group consisting of glutamic acid, lysine, tyrosine, threonine,phenylalanine, leucine, isoleucine, tryptophan, methionine, valine,alanine, histidine and arginine, and wherein the asparagine residuescorresponding to positions 21 and 52 in SEQ ID NO:4-7 (positions 13 and44 of SEQ ID NO 51 or 52) are conserved.

A number of the Fc-binding domains listed above are shown aligned inFIG. 1. The parental, i.e. non-engineered, Staphylococcus Protein A(SpA) comprises five Fc-dining domains termed domain E (SEQ ID NO 1), D(SEQ ID NO 2), A (SEQ ID NO 3), B (SEQ ID NO 4) and C (SEQ ID NO 5).Protein Z (SEQ ID NO:6) is a mutated B-domain as disclosed in U.S. Pat.No. 5,143,844, hereby incorporated by reference in its entirety. SEQ IDNO 7 denotes a further mutated variant of Protein Z, here called Zvar,with the mutations N3A,N6D,N23T. SEQ ID NO:22 (not shown in FIG. 1) is anatural variant of the A-domain in Protein A from Staphylococcus aureusstrain N315, having an A46S mutation, using the position terminology ofFIG. 1. SEQ ID NO 51 is Zvar (SEQ ID NO 7) without the linker regionamino acids at positions 1-8 and 56-58. SEQ ID NO 52 is the C-domain ofprotein A without the linker region amino acids 1-8 and 56-58) asillustrated in FIG. 1.

The mutation of N11 (N3 of SEQ ID NO 51:52) in these domains, togetherwith the conservation of the asparagine residues N21 and N52 (N13 andN44 of SEQ ID NO 51:52) confers an improved alkali stability incomparison with the parental domain/polypeptide, without impairing theimmunoglobulin-binding properties. Hence, the polypeptide can also bedescribed as an Fc- or immunoglobulin-binding polypeptide, oralternatively as an Fc- or immunoglobulin-binding polypeptide unit.

Described in alternative language, the immunoglobulin-bindingalkali-stabilized Protein A domains may comprise, consist essentiallyof, or consist of a sequence as defined by, or having at least 90%, atleast 95% or at least 98% identity to SEQ ID NO 53.

SEQ ID NO 53 X₁Q X₂AFYEILX₃LP NLTEEQRX₄X₅F IX₆X₇LKDX₈PSX₉SX₁₀X₁₁X₁₂LAEAKX₁₃ X₁₄NX₁₅AQ.wherein individually of each other:X₁=A, Q or is deleted

X₂=E,K,Y,T,F,L,W,I,M,V,A,H or R X₃=H or K X₄=A or N

X₅=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, such asS,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K

X₆=Q or E X₇=S or K X₈=E or D

X₉=Q, V or is deletedX₁₀=K, R, A or is deletedX₁₁=A, E, N or is deleted

X₁₂=I or L X₁₃=K or R X₁₄=L or Y X₁₅=D, F,Y,W,K or R

Specifically, the amino acid residues in SEQ ID NO 53 may individuallyof each other be:

X₁=A or is deleted

X₂=E X₃=H X₄=N X₆=Q X₇=S X₈=D

X₉=V or is deletedX₁₀=K or is deletedX₁₁=A or is deleted

X₁₂=I X₁₃=K X₁₄=L

In certain embodiments, the amino acid residues in SEQ ID NO 53 may be:

X₁=A, X₂=E, X₃=H, X₄=N, X₆=Q, X₇=5, X₈=D, X₉=V, X₁₀=K, X₁₁=A, X₁₂=I,X₁₃=K, X₁₄=L.

In some embodiments X₂=E, X₃=H, X₄=N, X₅=A, X₆=Q, X₇=S, X₈=D, X₁₂=I,X₁₃=K, X₁₄=L and X₁₅=D and one or more of X₁, X₉, X₁₀ and X₁₁ isdeleted. In further embodiments, X₁=A, X₂=E, X₃=H, X₄=N,X₅=S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X₆=Q, X₇=S, X₈=D, X₉=V, X₁₀=K,X₁₁=A, X₁₂=I, X₁₃=K, X₁₄=L and X₁₅=D, or alternatively X₁=A, X₂=E, X₃=H,X₄=N, X₅=A, X₆=Q, X₇=S, X₈=D, X₉=V, X₁₀=K, X₁₁=A, X₁₂=I, X₁₃=K, X₁₄=LandX₁₅=F,Y,W,K or R.

In some embodiments, the amino acid residues may individually of eachother be:

a) X₁=A or is deleted, X₂=E, X₃=H, X₄=N, X₆=Q, X₇=5, X₈=D, X₉=V or isdeleted, X₁₀=K or is deleted, X₁₁=A or is deleted, X₁₂=I, X₁₃=K, X₁₄=L;b) X₁=A, X₂=E, X₃=H, X₄=N, X₅=A, X₆=Q, X₇=S, X₈=D, X₉=V, X₁₀=K, X₁₁=A,X₁₂=I, X₁₃=K, X₁₄=L and X₁₅=D;C) X₁ is A, X₂=E, X₃=H, X₄=N, X₆=Q, X₇=S, X₈=D, X₉=V, X₁₀=K, X₁₁=A,X₁₂=I, X₁₃=K, X₁₄=L and X₁₅=D; ord) X₁ is A, X₃=H, X₄=N, X₅=A, X₆=Q, X₇=S, X₈=D, X₉=V, X₁₀=K, X₁₁=A,X₁₂=I, X₁₃=K, X₁₄=L and X₁₅=D.

The N11 (X₂) mutation (e.g. a N11E or N11K mutation) may be the onlymutation or the polypeptide may also comprise further mutations, such assubstitutions in at least one of the positions corresponding topositions 3, 6, 9, 10, 15, 18, 23, 28, 29, 32, 33, 36, 37, 40, 42, 43,44, 47, 50, 51, 55 and 57 in SEQ ID NO:4-7. In one or more of thesepositions, the original amino acid residue may e.g. be substituted withan amino acid which is not asparagine, proline or cysteine. The originalamino acid residue may e.g. be substituted with an alanine, a valine, athreonine, a serine, a lysine, a glutamic acid or an aspartic acid.Further, one or more amino acid residues may be deleted, e.g. frompositions 1-6 and/or from positions 56-58.

In some embodiments, the amino acid residue at the positioncorresponding to position 9 in SEQ ID NO:4-7 (X₁) is an amino acid otherthan glutamine, asparagine, proline or cysteine, such as an alanine orit can be deleted. The combination of the mutations at positions 9 and11 provides particularly good alkali stability, as shown by theexamples. In specific embodiments, in SEQ ID NO: 7 the amino acidresidue at position 9 is an alanine and the amino acid residue atposition 11 is a lysine or glutamic acid, such as a lysine. Mutations atposition 9 are also discussed in copending applicationPCT/SE2014/050872, which is hereby incorporated by reference in itsentirety.

In some embodiments, the amino acid residue at the positioncorresponding to position 50 in SEQ ID NO:4-7 (X₁₃) is an arginine or aglutamic acid.

In certain embodiments, the amino acid residue at the positioncorresponding to position 3 in SEQ ID NO:4-7 is an alanine and/or theamino acid residue at the position corresponding to position 6 in SEQ IDNO:4-7 is an aspartic acid. One of the amino acid residues at positions3 and 6 may be an asparagine and in an alternative embodiment both aminoacid residues at positions 3 and 6 may be asparagines.

In some embodiments the amino acid residue at the position correspondingto position 43 in SEQ ID NO:4-7 (X₁₁) is an alanine or a glutamic acid,such as an alanine or it can be deleted. In specific embodiments, theamino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanineand lysine/glutamic acid respectively, while the amino acid residue atposition 43 is alanine or glutamic acid.

In certain embodiments the amino acid residue at the positioncorresponding to position 28 in SEQ ID NO:4-7 (X₅) is an alanine or anasparagine, such as an alanine.

In some embodiments the amino acid residue at the position correspondingto position 40 in SEQ ID NO:4-7 (X₉) is selected from the groupconsisting of asparagine, alanine, glutamic acid and valine, or from thegroup consisting of glutamic acid and valine, or valine, or it can bedeleted. In specific embodiments, the amino acid residues at positions 9and 11 in SEQ ID NO: 7 are alanine and glutamic acid respectively, whilethe amino acid residue at position 40 is valine. Optionally, the aminoacid residue at position 43 may then be alanine or glutamic acid.

In certain embodiments, the amino acid residue at the positioncorresponding to position 42 in SEQ ID NO:4-7 (X₁₀) is an alanine,lysine or arginine or it can be deleted.

In some embodiments the amino acid residue at the position correspondingto position 18 in SEQ ID NO:4-7 (X₃) is a lysine or a histidine, such asa lysine.

In certain embodiments the amino acid residue at the positioncorresponding to position 33 in SEQ ID NO:4-7 (X₇) is a lysine or aserine, such as a lysine.

In some embodiments the amino acid residue at the position correspondingto position 37 in SEQ ID NO:4-7 (X₈) is a glutamic acid or an asparticacid, such as a glutamic acid.

In certain embodiments the amino acid residue at the positioncorresponding to position 51 in SEQ ID NO:4-7 (X₁₄) is a tyrosine or aleucine, such as a tyrosine.

In some embodiments, the amino acid residue at the positioncorresponding to position 44 in SEQ ID NO:4-7 (X₁₂) is a leucine or anisoleucine. In specific embodiments, the amino acid residues atpositions 9 and 11 in SEQ ID NO: 7 are alanine and lysine/glutamic acidrespectively, while the amino acid residue at position 44 is isoleucine.Optionally, the amino acid residue at position 43 may then be alanine orglutamic acid.

In some embodiments, the amino acid residues at the positionscorresponding to positions 1, 2, 3 and 4 or to positions 3, 4, 5 and 6in SEQ ID NO: 4-7 have been deleted. In specific variants of theseembodiments, the parental polypeptide is the C domain of Protein A (SEQID NO: 5). The effects of these deletions on the native C domain aredescribed in U.S. Pat. Nos. 9,018,305 and 8,329,860, which are herebyincorporated by reference in their entireties.

In certain embodiments, the mutation in SEQ ID NO 4-7, such as in SEQ IDNO 7, is selected from the group consisting of: N11K; N11E; N11Y; N11T;N11F; N11L; N11W; N11I; N11M; N11V; N11A; N11H; N11R; N11E,Q32A;N11E,Q32E,Q40E; N11E,Q32E,K50R; Q9A,N11E,N43A; Q9A,N11E,N28A,N43A;Q9A,N11E,Q40V,A42K,N43E,L44I; Q9A,N11E,Q40V,A42K,N43A,L44I;N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y;Q9A,N11E,N28A,Q40V,A42K,N43A,L44I;Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y; N11K, H18K, D37E,A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I; Q9A, N11K,H18K, D37E, A42R, N43A, L44I, K50R; Q9A,N11K,H18K,D37E,A42R;Q9A,N11E,D37E,Q40V,A42K,N43A,L44I and Q9A,N11E,D37E,Q40V,A42R,N43A,L44I.These mutations provide particularly high alkaline stabilities. Themutation in SEQ ID NO 4-7, such as in SEQ ID NO 7, can also be selectedfrom the group consisting of N11K; N11Y; N11F; N11L; N11W; N11I; N11M;N11V; N11A; N11H; N11R; Q9A,N11E,N43A; Q9A,N11E,N28A,N43A;Q9A,N11E,Q40V,A42K,N43E,L44I; Q9A,N11E,Q40V,A42K,N43A,L44I;Q9A,N11E,N28A,Q40V,A42K,N43A,L44I;N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y;Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y; N11K, H18K, D37E,A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I and Q9A, N11K,H18K, D37E, A42R, N43A, L44I, K50R.

In some embodiments, the polypeptide comprises or consists essentiallyof a sequence defined by or having at least 90%, 95% or 98% identity toan amino acid sequence selected from the group consisting of: SEQ ID NO8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13,SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24,SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29,SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40,SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45,SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49 and SEQ ID NO 50.It may e.g. comprise or consist essentially of a sequence defined by orhaving at least 90%, 95% or 98% identity to an amino acid sequenceselected from the group consisting of: SEQ ID NO 8, SEQ ID NO 9, SEQ IDNO 10, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28 and SEQ ID NO 29. It canalso comprise or consist essentially of a sequence defined by or havingat least 90%, 95% or 98% identity to an amino acid sequence selectedfrom the group consisting of: SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10,SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25,SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 38, SEQ ID NO 40; SEQ ID NO 41;SEQ ID NO 42; SEQ NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQID NO 47 and SEQ ID NO 48.

In certain embodiments, the polypeptide comprises or consistsessentially of a sequence defined by or having at least 90%, 95% or 98%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO 54-70; comprises or consists essentially of a sequence definedby or having at least 90%, 95% or 98% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO 71-75; or it maycomprise or consist essentially of a sequence defined by or having atleast 90%, 95% or 98% identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO 76-79. It may further comprise orconsist essentially of a sequence defined by or having at least 90%, 95%or 98% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO 89-95.

The polypeptide may e.g. be defined by a sequence selected from thegroups above or from subsets of these groups, but it may also compriseadditional amino acid residues at the N- and/or C-terminal end, e.g. aleader sequence at the N-terminal end and/or a tail sequence at theC-terminal end.

SEQ ID NO 8 Zvar(Q9A, N11E, N43A)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKSEQ ID NO 9 Zvar(Q9A, N11E, N28A, N43A)VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKSEQ ID NO 10 Zvar(Q9A, N11E, Q40V, A42K, N43E, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPKSEQ ID NO 11 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 12 Zvar(N11E, Q32A)VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IASLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 13 Zvar(N11E)VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 14 Zvar(N11E, Q32E, Q40E)VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSE SANLLAEAKK LNDAQAPKSEQ ID NO 15 Zvar(N11E, Q32E, K50R)VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSQ SANLLAEAKR LNDAQAPKSEQ ID NO 16 Zvar(N11K)VDAKFDKEQQ KAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 23 Zvar(N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y)VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKSEQ ID NO 24 Zvar(Q9A, N11E, N28A, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 25Zvar(Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y)VDAKFDKEAQ KAFYEILKLP NLTEEQRAAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKSEQ ID NO 26 Zvar(N11K, H18K, D37E, A42R, N43A, L44I)VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKK LNDAQAPKSEQ ID NO 27 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I)VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKK LNDAQAPKSEQ ID NO 28 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKR LNDAQAPKSEQ ID NO 29 Zvar(Q9A, N11K, H18K, D37E, A42R)VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKSEQ ID NO 36 B(Q9A, N11E, Q40V, A42K, N43A, L44I)ADNKFNKEAQ EAFYEILHLP NLNEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 37 C(Q9A, N11E, E43A)ADNKFNKEAQ EAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 38 Zvar(N11Y)VDAKFDKEQQ YAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAN LLAEAKK LNDAQAPKSEQ ID NO 39 Zvar(N11T)VDAKFDKEQQ TAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAN LLAEAKK LNDAQAPKSEQ ID NO 40 Zvar(N11F)VDAKFDKEQQ FAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAN LLAEAKK LNDAQAPKSEQ ID NO 41 Zvar(N11L)VDAKFDKEQQ LAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAN LLAEAKK LNDAQAPKSEQ ID NO 42 Zvar(N11W)VDAKFDKEQQ WAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 43 Zvar(N11I)VDAKFDKEQQ IAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAN LLAEAKK LNDAQAPKSEQ ID NO 44 Zvar(N11M)VDAKFDKEQQ MAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAN LLAEAKK LNDAQAPKSEQ ID NO 45 Zvar(N11V)VDAKFDKEQQ VAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 46 Zvar(N11A)VDAKFDKEQQ AAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 47 Zvar(N11H)VDAKFDKEQQ HAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 48 Zvar(N11R)VDAKFDKEQQ RAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPKSEQ ID NO 49 Zvar(Q9A, N11E, D37E, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPKSEQ ID NO 50 Zvar(Q9A, N11E, D37E, Q40V, A42R, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPKSEQ ID NO 54 Zvar(Q9A, N11E, A29G, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 55 Zvar(Q9A, N11E, A295, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNSF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 56 Zvar(Q9A, N11E, A29Y, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNYF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 57 Zvar(Q9A, N11E, A29Q, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNQF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 58 Zvar(Q9A, N11E, A29T, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNTF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 59 Zvar(Q9A, N11E, A29N, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNNF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 60 Zvar(Q9A, N11E, A29F, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNFF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 61 Zvar(Q9A, N11E, A29L, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNLF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 62 Zvar(Q9A, N11E, A29W, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNWF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 63 Zvar(Q9A, N11E, A291, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNIF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 64 Zvar(Q9A, N11E, A29M, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNMF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 65 Zvar(Q9A, N11E, A29V, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNVF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 66 Zvar(Q9A, N11E, A29D, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNDF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 67 Zvar(Q9A, N11E, A29E, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNEF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 68 Zvar(Q9A, N11E, A29H, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNHF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 69 Zvar(Q9A, N11E, A29R, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNRF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 70 Zvar(Q9A, N11E, A29K, Q40V, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNKF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 71 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53F)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNFAQAPKSEQ ID NO 72 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53Y)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNYAQAPKSEQ ID NO 73 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53W)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNWAQAPKSEQ ID NO 74 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53K)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNKAQAPKSEQ ID NO 75 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53R)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNRAQAPKSEQ ID NO 76 Zvar(Q9del, N11E, Q40V, A42K, N43A, L44I)VDAKFDKE_Q EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 77 Zvar(Q9A, N11E, Q40del, A42K, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPS_ SKAILAEAKK LNDAQAPKSEQ ID NO 78 Zvar(Q9A, N11E, Q40V, A42del, N43A, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV S_AILAEAKK LNDAQAPKSEQ ID NO 79 Zvar(Q9A, N11E, Q40V, A42K, N43del, L44I)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SK_ILAEAKK LNDAQAPKSEQ ID NO 89Zvar(D2del, A3del, K4del, Q9A, N11E, Q40V, A42K, N43A, L44I)V___FDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 90Zvar(V1del, D2del, Q9A, N11E, Q40V, A42K, N43A, L44I, K58del)__AKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAP_SEQ ID NO 91Zvar(K4del, F5del, D6del, K7del, E8del, Q9A, N11E, Q40V, A42K, N43A, L44I)VDA_____AQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 92Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, A56del, P57del, K58del)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQ___SEQ ID NO 93Zvar(V1del,, D2del, A3del, Q9A, N11E, Q40V, A42K, N43A, L44I)___KFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 94Zvar(V1del, D2del, A3del, K4del, F5del, D6del, K7del, E8del, Q9A, N11E, Q40V, A42K, N43A, L44I)________AQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKSEQ ID NO 95 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, K58_insYEDG)VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKYE DG

The separation matrix comprises multimers of the immunoglobulin-bindingalkali-stabilized Protein A domains. Such multimers comprise, consistessentially of, or consist of a plurality of immunoglobulin-bindingalkali-stabilized Protein A domains (polypeptide units) as defined byany embodiment disclosed above. The use of multimers may increase theimmunoglobulin binding capacity and multimers may also have a higheralkali stability than monomers. The multimer can e.g. be a dimer, atrimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer or anonamer. The multimer may be a homomultimer, where all the units in themultimer are identical or it can be a heteromultimer, where at least oneunit differs from the others. Advantageously, all the units in themultimer are alkali stable, such as by comprising themutations/conservations disclosed above. The polypeptides can be linkedto each other directly by peptide bonds between the C-terminal andN-terminal ends of the polypeptides. Alternatively, two or more units inthe multimer can be linked by linkers comprising oligomeric or polymericspecies, such as linkers comprising peptides with up to 25 or 30 aminoacids, such as 3-25 or 3-20 amino acids. The linkers may e.g. compriseor consist essentially of a peptide sequence defined by, or having atleast 90% identity or at least 95% identity, with an amino acid sequenceselected from the group consisting of APKVDAKFDKE, APKVDNKFNKE,APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE,APK, APKYEDGVDAKFDKE and YEDG or alternatively selected from the groupconsisting of APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE,APKKFDKE, APKYEDGVDAKFDKE and YEDG. They can also consist essentially ofa peptide sequence defined by or having at least 90% identity or atleast 95% identity with an amino acid sequence selected from the groupconsisting of APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE,APKKFDKE, APK and APKYEDGVDAKFDKE. In some embodiments the linkers donot consist of the peptides APKVDAKFDKE or APKVDNKFNKE, or alternativelydo not consist of the peptides APKVDAKFDKE, APKVDNKFNKE, APKFNKE,APKFDKE, APKVDKE or APKADKE.

The nature of such a linker should preferably not destabilize thespatial conformation of the protein units. This can e.g. be achieved byavoiding the presence of proline in the linkers. Furthermore, saidlinker should preferably also be sufficiently stable in alkalineenvironments not to impair the properties of the mutated protein units.For this purpose, it is advantageous if the linkers do not containasparagine. It can additionally be advantageous if the linkers do notcontain glutamine. The multimer may further at the N-terminal endcomprise a plurality of amino acid residues e.g. originating from thecloning process or constituting a residue from a cleaved off signalingsequence. The number of additional amino acid residues may e.g. be 20 orless, such as 15 or less, such as 10 or less or 5 or less. As a specificexample, the multimer may comprise an AQ, AQGT, VDAKFDKE, AQVDAKFDKE orAQGTVDAKFDKE sequence at the N-terminal end.

In certain embodiments, the multimer may comprise, or consistessentially, of a sequence selected from the group consisting of: SEQ IDNO 80-87. These and additional sequences are listed below and named asParent(Mutations)n, where n is the number of monomer units in amultimer.

SEQ ID NO 17 Zvar(Q9A, N11E, N43A)4AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKC SEQ ID NO 18Zvar(Q9A, N11E, N28A, N43A)4AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKC SEQ ID NO 19Zvar(Q9A, N11E, Q40V, A42K, N43E, L44I)4AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPKC SEQ ID NO 20Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)4AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 30Zvar(N11K, H18K, 533K, D37E, A42R, N43A, L44I, K50R, L51Y)4AQGT VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKVDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKVDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKVDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKCSEQ ID NO 31 Zvar(Q9A, N11K, H18K, D37E, A42R)4AQGT VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKVDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKVDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKVDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKCSEQ ID NO 32 Zvar(Q9A, N11E, N28A, Q40V, A42K, N43A, L44I)4AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 33Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)6AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAFIQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSVSKAILAEAKK LNDAQAPKC SEQ ID NO 34Zvar(Q9A, N11E, D37E, Q40V, A42K, N43A, L44I)4AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 35Zvar(Q9A, N11E, D37E, Q40V, A42R, N43A, L44I)4AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPKC SEQ ID NO 80Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with D2, A3 and K4 in linker deletedVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 81Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with K58, V1 and D2 in linker deletedVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAP AKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 82Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with P57, K58, V1, D2 and A3 in linkerdeletedVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAP AKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 83Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with K4, F5, D6, K7 and E8 in linkerdeletedVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 84Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with A56, P57 and K58 in linker deletedVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQ VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 85Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with V1, D2 and A3 in linker deletedVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK KFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 86Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with V1, D2, A3, K4, F5, D6, K7 and E8 inlinker deletedVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK AQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC SEQ ID NO 87Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)2 with YEDG inserted in linker betweenK58 and V1VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK YEDGVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKCSEQ ID NO 88 Zvar2VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

In some embodiments, the polypeptide and/or multimer, as disclosedabove, further comprises at the C-terminal or N-terminal end one or morecoupling elements, selected from the group consisting of one or morecysteine residues, a plurality of lysine residues and a plurality ofhistidine residues. The coupling element(s) may also be located within1-5 amino acid residues, such as within 1-3 or 1-2 amino acid residuesfrom the C-terminal or N-terminal end. The coupling element may e.g. bea single cysteine at the C-terminal end. The coupling element(s) may bedirectly linked to the C- or N-terminal end, or it/they may be linkedvia a stretch comprising up to 15 amino acids, such as 1-5, 1-10 or 5-10amino acids. This stretch should preferably also be sufficiently stablein alkaline environments not to impair the properties of the mutatedprotein. For this purpose, it is advantageous if the stretch does notcontain asparagine. It can additionally be advantageous if the stretchdoes not contain glutamine. An advantage of having a C-terminal cysteineis that endpoint coupling of the protein can be achieved throughreaction of the cysteine thiol with an electrophilic group on a support.This provides excellent mobility of the coupled protein which isimportant for the binding capacity.

The alkali stability of the polypeptide or multimer can be assessed bycoupling it to a surface plasmon resonance (SPR) chip, e.g. to BiacoreCM5 sensor chips as described in the examples, using e.g. NHS- ormaleimide coupling chemistries, and measuring the immunoglobulin-bindingcapacity of the chip, typically using polyclonal human IgG, before andafter incubation in alkaline solutions at a specified temperature, e.g.22+/−2° C. The incubation can e.g. be performed in 0.5 M NaOH for anumber of 10 min cycles, such as 100, 200 or 300 cycles. The IgGcapacity of the matrix after 100 10 min incubation cycles in 0.5 M NaOHat 22+/−2° C. can be at least 55, such as at least 60, at least 80 or atleast 90% of the IgG capacity before the incubation. Alternatively, theremaining IgG capacity after 100 cycles for a particular mutant measuredas above can be compared with the remaining IgG capacity for theparental polypeptide/multimer. In this case, the remaining IgG capacityfor the mutant may be at least 105%, such as at least 110%, at least125%, at least 150% or at least 200% of the parentalpolypeptide/multimer.

The immunoglobulin-binding alkali-stabilized Protein A domains and/ormultimers thereof may be encoded by a nucleic acid sequence, such as anRNA sequence or a DNA sequence encoding the polypeptide or multimer. Avector, such as a plasmid, which in addition to the coding sequencecomprises the required signal sequences, may be used for expression ofthe polypeptide or multimer. The vector may comprise nucleic acidencoding a multimer as described above, wherein the separate nucleicacids encoding each unit may have homologous or heterologous DNAsequences.

An expression system, which comprises a nucleic acid or a vector asdisclosed above, may be used for expression of the polypertide ormultimer. The expression system may e.g. be a gram-positive orgram-negative prokaryotic host cell system, e.g. E. coli or Bacillus sp.which has been modified to express the present polypeptide or multimer.Alternatively, the expression system may be a eukaryotic host cellsystem, such as a yeast, e.g. Pichia pastoris or Saccharomycescerevisiae, or mammalian cells, e.g. CHO cells.

The separation matrix comprises, consists essentially of, or consists ofmultimers of immunoglobulin-binding alkali-stabilized Protein A domainsas described above, covalently coupled to a porous support.

The separation matrix may comprise at least 11, such as 11-21, 15-21 or15-18 mg/ml Fc-binding ligands covalently coupled to a porous support,wherein:

a) the ligands comprise multimers of alkali-stabilized Protein Adomains,b) the porous support comprises cross-linked polymer particles having avolume-weighted median diameter (d50,v) of 56-70, such as 56-66,micrometers and a dry solids weight of 55-80, such as 60-78 or 65-78,mg/ml. The cross-linked polymer particles may further have a pore sizecorresponding to an inverse gel filtration chromatography Kd value of0.69-0.85, such as 0.70-0.85 or 0.69-0.80, for dextran of Mw 110 kDa.The multimers may e.g. comprise tetramers, pentamers, hexamers orheptamers of alkali-stabilized Protein A domains, such as hexamers ofalkali-stabilized Protein A domains. The combination of the high ligandcontents with the particle size range, the dry solids weight range andthe optional Kd range provides for a high binding capacity, e.g. suchthat the 10% breakthrough dynamic binding capacity for IgG is at least45 mg/ml, such as at least 50 or at least 55 mg/ml at 2.4 min residencetime. Alternatively, or additionally, the 10% breakthrough dynamicbinding capacity for IgG may be at least 60 mg/ml, such as at least 65,at least 70 or at least 75 mg/ml at 6 min residence time.

The alkali-stabilized Protein A domain multimers are highly selectivefor IgG and the separation matrix can suitably have a dissociationconstant for human IgG2 of below 0.2 mg/ml, such as below 0.1 mg/ml, in20 mM phosphate buffer, 180 mM NaCl, pH 7.5. This can be determinedaccording to the adsorption isotherm method described in N Pakiman etal: J Appl Sci 12, 1136-1141 (2012).

In certain embodiments the separation matrix comprises at least 15, suchas 15-21 or 15-18 mg/ml Fc-binding ligands covalently coupled to aporous support, wherein the ligands comprise multimers ofalkali-stabilized Protein A domains. These multimers can suitably be asdisclosed in any of the embodiments described above or as specifiedbelow.

In some embodiments the separation matrix comprises 5-25, such as 5-20mg/ml, 5-15 mg/ml, 5-11 mg/ml or 6-11 mg/ml of the polypeptide ormultimer coupled to the support. The amount of coupledpolypeptide/multimer can be controlled by the concentration ofpolypeptide/multimer used in the coupling process, by the activation andcoupling conditions used and/or by the pore structure of the supportused. As a general rule the absolute binding capacity of the matrixincreases with the amount of coupled polypeptide/multimer, at least upto a point where the pores become significantly constricted by thecoupled polypeptide/multimer. Without being bound by theory, it appearsthough that for the Kd values recited for the support, the constrictionof the pores by coupled ligand is of lower significance. The relativebinding capacity per mg coupled polypeptide/multimer will decrease athigh coupling levels, resulting in a cost-benefit optimum within theranges specified above.

Such a separation matrix is useful for separation of immunoglobulins orother Fc-containing proteins and, due to the improved alkali stabilityof the polypeptides/multimers, the matrix will withstand highly alkalineconditions during cleaning, which is essential for long-term repeateduse in a bioprocess separation setting. The alkali stability of thematrix can be assessed by measuring the immunoglobulin-binding capacity,typically using polyclonal human IgG, before and after incubation inalkaline solutions at a specified temperature, e.g. 22+/−2° C. Theincubation can e.g. be performed in 0.5 M or 1.0 M NaOH for a number of15 min cycles, such as 100, 200 or 300 cycles, corresponding to a totalincubation time of 25, 50 or 75 h. The IgG capacity of the matrix after96-100 15 min incubation cycles or a total incubation time of 24 or 25 hin 0.5 M NaOH at 22+/−2° C. can be at least 80, such as at least 85, atleast 90 or at least 95% of the IgG capacity before the incubation. Thecapacity of the matrix after a total incubation time of 24 h in 1.0 MNaOH at 22+/−2° C. can be at least 70, such as at least 80 or at least90% of the IgG capacity before the incubation. The the 10% breakthroughdynamic binding capacity (Qb10%) for IgG at 2.4 min or 6 min residencetime may e.g. be reduced by less than 20% after incubation 31 h in 1.0 Maqueous NaOH at 22+/−2 C.

As the skilled person will understand, the expressed polypeptide ormultimer should be purified to an appropriate extent before beingimmobilized to a support. Such purification methods are well known inthe field, and the immobilization of protein-based ligands to supportsis easily carried out using standard methods. Suitable methods andsupports will be discussed below in more detail.

The porous support of the separation matrix may be of any suitablewell-known kind. A conventional affinity separation matrix is often oforganic nature and based on polymers that expose a hydrophilic surfaceto the aqueous media used, i.e. expose hydroxy (—OH), carboxy (—COOH),carboxamido (—CONH₂, possibly in N-substituted forms), amino (—NH₂,possibly in substituted form), oligo- or polyethylenoxy groups on theirexternal and, if present, also on internal surfaces. The porosity of thesupport can be expressed as a Kay or Kd value (the fraction of the porevolume available to a probe molecule of a particular size) measured byinverse size exclusion chromatography, e.g. according to the methodsdescribed in Gel Filtration Principles and Methods, Pharmacia LKBBiotechnology 1991, pp 6-13. Kay is determined as the ratio(V_(e)−V₀)/(V_(t)−V₀), where Ve is the elution volume of a probemolecule (e.g. Dextran 110 kD), V₀ is the void volume of the column(e.g. the elution volume of a high Mw void marker, such as raw dextran)and V_(t) is the total volume of the column. Kd can be determined as(V_(e)—V₀)/V_(i), where V_(i) is the elution volume of a salt (e.g.NaCl) able to access all the volume except the matrix volume (the volumeoccupied by the matrix polymer molecules). By definition, both Kd andKay values always lie within the range 0-1. The Kay value canadvantageously be 0.6-0.95, e.g. 0.7-0.90 or 0.6-0.8, as measured withdextran of Mw 110 kDa as a probe molecule. The Kd value as measured withdextran of Mw 110 kDa can suitably be 0.68-0.90, such as 0.68-0.85 or0.70-0.85. An advantage of this is that the support has a large fractionof pores able to accommodate both the polypeptides/multimers of theinvention and immunoglobulins binding to the polypeptides/multimers andto provide mass transport of the immunoglobulins to and from the bindingsites.

The polypeptides or multimers may be attached to the porous support viaconventional coupling techniques utilising e.g. thiol, amino and/orcarboxy groups present in the ligand. Bisepoxides, epichlorohydrin,CNBr, N-hydroxysuccinimide (NHS) etc are well-known coupling reagents.Between the support and the polypeptide/multimer, a molecule known as aspacer can be introduced, which improves the availability of thepolypeptide/multimer and facilitates the chemical coupling of thepolypeptide/multimer to the support. Depending on the nature of thepolypeptide/multimer and the coupling conditions, the coupling may be amultipoint coupling (e.g. via a plurality of lysines) or a single pointcoupling (e.g. via a single cysteine).

In certain embodiments the polypeptides or multimers are coupled to thesupport via thioether bonds. Methods for performing such coupling arewell-known in this field and easily performed by the skilled person inthis field using standard techniques and equipment. Thioether bonds areflexible and stable and generally suited for use in affinitychromatography. In particular when the thioether bond is via a terminalor near-terminal cysteine residue on the polypeptide or multimer, themobility of the coupled polypeptide/multimer is enhanced which providesimproved binding capacity and binding kinetics. In some embodiments thepolypeptide/multimer is coupled via a C-terminal cysteine provided onthe protein as described above. This allows for efficient coupling ofthe cysteine thiol to electrophilic groups, e.g. epoxide groups,halohydrin groups etc. on a support, resulting in a thioether bridgecoupling.

In certain embodiments the support comprises a polyhydroxy polymer, suchas a polysaccharide. Examples of polysaccharides include e.g. dextran,starch, cellulose, pullulan, agar, agarose etc. Polysaccharides areinherently hydrophilic with low degrees of nonspecific interactions,they provide a high content of reactive (activatable) hydroxyl groupsand they are generally stable towards alkaline cleaning solutions usedin bioprocessing.

In some embodiments the support comprises agar or agarose. The supportsused in the present invention can easily be prepared according tostandard methods, such as inverse suspension gelation (S Hjertén:Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the basematrices are commercially available products, such as crosslinkedagarose beads sold under the name of SEPHAROSE™ FF (GE Healthcare). Inan embodiment, which is especially advantageous for large-scaleseparations, the support has been adapted to increase its rigidity usingthe methods described in U.S. Pat. No. 6,602,990 or 7,396,467, which arehereby incorporated by reference in their entireties, and hence rendersthe matrix more suitable for high flow rates.

In certain embodiments the support, such as a polymer, polysaccharide oragarose support, is crosslinked, such as with hydroxyalkyl ethercrosslinks. Crosslinker reagents producing such crosslinks can be e.g.epihalohydrins like epichlorohydrin, diepoxides like butanedioldiglycidyl ether, allylating reagents like allyl halides or allylglycidyl ether. Crosslinking is beneficial for the rigidity of thesupport and improves the chemical stability. Hydroxyalkyl ethercrosslinks are alkali stable and do not cause significant nonspecificadsorption.

Alternatively, the porous support is based on synthetic polymers, suchas polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides etc. In case ofhydrophobic polymers, such as matrices based on divinyl andmonovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilised to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers are easily produced accordingto standard methods, see e.g. “Styrene based polymer supports developedby suspension polymerization” (R Arshady: Chimica e L'Industria 70(9),70-75 (1988)). Alternatively, a commercially available product, such asSOURCE™ (GE Healthcare) is used. In another alternative, the poroussupport according to the invention comprises a support of inorganicnature, e.g. silica, zirconium oxide etc.

In yet another embodiment, the solid support is in another form such asa surface, a chip, capillaries, or a filter (e.g. a membrane or a depthfilter matrix).

As regards the shape of the matrix according to the invention, in oneembodiment the matrix is in the form of a porous monolith. In analternative embodiment, the matrix is in beaded or particle form thatcan be porous or non-porous. Matrices in beaded or particle form can beused as a packed bed or in a suspended form. Suspended forms includethose known as expanded beds and pure suspensions, in which theparticles or beads are free to move. In case of monoliths, packed bedand expanded beds, the separation procedure commonly followsconventional chromatography with a concentration gradient. In case ofpure suspension, batch-wise mode will be used.

The separation matrix as disclosed above has excellent alkali stabilityand may be stored in an alkaline storage liquid. The method of storingthe separation matrix comprises the following steps:

-   -   a) providing a storage liquid comprising at least 50% by volume        of an aqueous alkali metal hydroxide solution;    -   b) permeating the separation matrix with the storage liquid; and    -   c) storing the storage liquid-permeated separation matrix for a        storage time of at least 5 days.

By using a storage liquid comprising aqueous alkali metal hydroxidesolution, a bacteriostatic or bactericidal solution may be obtainedwithout requiring the use of alcohols such as ethanol, isopropanol, orbenzyl alcohol. This means that a storage liquid may be used that ischeaper, subject to less regulatory burden, non-flammable and easier todispose of than the storage solutions presently used for known protein Aaffinity separation matrices.

The separation matrix is a separation matrix as disclosed above,comprising multimers of immunoglobulin-binding alkali-stabilized ProteinA domains covalently coupled to a porous support, wherein thealkali-stabilized Protein A domains comprise mutants of a parentalFc-binding domain of Staphylococcus Protein A (SpA), as defined by, orhaving at least 80% such as at least 90%, 95% or 98% identity to, SEQ IDNO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13and 44 of SEQ ID NO 51 or 52 are asparagines, and wherein at least theasparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutatedto an amino acid selected from the group consisting of glutamic acid,lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine,tryptophan, methionine, valine, alanine, histidine and arginine. Theimmunoglobulin-binding alkali-stabilized Protein A domains may comprisefurther mutations. For example, the glutamine residue at position 1 ofSEQ ID NO 51 or 52 may be mutated to an alanine; and/or the asparagineor glutamic acid residue at position 35 of SEQ ID NO 51 or 52 may bemutated to an alanine.

The storage liquid comprises at least 50% by volume of an aqueous alkalimetal hydroxide solution. The aqueous alkali metal hydroxide solutionmay comprise a single alkali metal hydroxide or a mixture of alkalimetal hydroxides, such as sodium hydroxide, potassium hydroxide, or amixture of sodium hydroxide and potassium hydroxide. The aqueous alkalimetal hydroxide solution may have a molarity of from 10 mM to 100 mM,such as from 30 mM to 50 mM, expressed as the total combinedconcentration of alkali metal hydroxides if a mixture of alkali metalhydroxides is used. The storage liquid may essentially consist of, orconsist of, the aqueous alkali metal hydroxide solution. However, thestorage liquid may in some embodiments also comprise further components.Such further components may include alcohols, such as a C₂-C₇ alcohol,such as ethanol, isopropanol or benzyl alcohol. Such further componentsmay include salts, such as sodium chloride. The use of alcohols and/orsalts in the storage liquid may increase the efficacy of the storageliquid in inhibiting or inactivating certain microorganisms, such asspore-forming bacteria.

Non-limiting examples of storage liquids include:

Sodium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M);Potassium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M);Sodium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M) with 10-20% byvolume ethanol;Sodium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M) with 10-50% byvolume isopropanol;Sodium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M) with 1-5% byvolume benzyl alcohol;Potassium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M) with 10-20%by volume ethanol;Potassium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M) with 10-50%by volume isopropanol; orPotassium hydroxide solution (0.01 M, 0.03 M, 0.05 M, 0.1 M) with 1-5%by volume benzyl alcohol.

The separation matrix is permeated with the storage liquid prior to andduring storage. By permeated with storage liquid, it is meant that thepores and interstices of the separation matrix are to a large extentfilled with storage liquid. The separation matrix should be permeatedwith a quantity of storage liquid sufficient to inhibit growth ofmicroorganisms in the stored separation matrix. The separation matrixmay be impregnated, saturated with, or immersed in the storage liquid.Typically, a slurry of separation matrix in storage liquid suitable forstorage may comprise about 50% to 80% by weight of separation matrix,relative to the total weight of the slurry.

The separation matrix may be stored in the storage liquid for asextended a period as required. Typically, if the separation matrix is tobe stored, this is for at least 5 days, often for at least 10 days, suchas at least 50 days, or such as at least 100 days, or such as at least200 days. The maximum storage time, or shelf life, of the separationmatrix depends on the nature of the storage liquid used, i.e. alkaliconcentration, as well as the degree of capacity loss acceptable to theuser, but may for example be up to 400 days or up to 700 days.

The mixture of storage liquid and separation matrix is contained in asuitable storage receptacle. The storage receptacle may be a bottle, canor drum made from a liquid-impervious material such as plastic, e.g.polyethylene, or glass. The separation matrix may be packaged in suchstorage receptacles for initial storage and distribution afterproduction, or may be re-filled into such storage receptacles after usein purifying an immunoglobulin. The storage receptacle may alternativelybe a pre-packed product for use in development or manufacturing ofimmunoglobulins. Such pre-packed products include filter plates, such as96-well filter plates, and pre-packed columns. Such pre-packed columnsinclude columns of all sizes known to the skilled person, fromlaboratory scale to process scale. Such columns can be shippedprepacked, qualified and sanitized, thus substantially reducing the timerequired for immunoglobulin purification processes.

The storage receptacle for storing the separation matrix may be open,vented or sealed. Since aqueous alkali metal hydroxide solutions arenon-flammable and relatively non-volatile, no special considerationsmust be given regarding pressure build-up in the storage receptacle andventilation of the storage room. In order to prevent dry-out of theseparation media, it is preferable if the storage media is stored in anairtight receptacle.

The separation matrix may be stored at any temperature known in the artfor storage of affinity media, such as from 1° C. to 30° C., or from 10to 20° C. However, prolonged storage at elevated temperatures maydegrade the capacity of the separation matrix, and therefore it ispreferable if the separation matrix can be stored at a temperature offrom 2 to 8° C.

If the separation matrix has been previously used for purifying animmunoglobulin prior to storage, e.g. if it has been used in aproduction campaign that has now concluded, it is preferable that theseparation matrix is cleaned and/or sanitized prior to storage. Cleaningliquids comprising at least 50% by volume of an aqueous alkali metalhydroxide solution and having a molarity of 0.5 M to 5 M may suitably beused to clean and/or sanitize the separation matrix.

EXAMPLES Mutagenesis of Protein

Site-directed mutagenesis was performed by a two-step PCR usingoligonucleotides coding for the mutations. As template a plasmidcontaining a single domain of either Z, B or C was used. The PCRfragments were ligated into an E. coli expression vector. DNA sequencingwas used to verify the correct sequence of inserted fragments.

To form multimers of mutants an Acc I site located in the startingcodons (GTA GAC) of the B, C or Z domain was used, corresponding toamino acids VD. The vector for the monomeric domain was digested withAcc I and phosphatase treated. Acc I sticky-ends primers were designed,specific for each variant, and two overlapping PCR products weregenerated from each template. The PCR products were purified and theconcentration was estimated by comparing the PCR products on a 2%agarose gel. Equal amounts of the pair wise PCR products were hybridized(90° C.->25° C. in 45 min) in ligation buffer. The resulting productconsists approximately to ¼ of fragments likely to be ligated into anAcc I site (correct PCR fragments and/or the digested vector). Afterligation and transformation colonies were PCR screened to identifyconstructs containing the desired mutant. Positive clones were verifiedby DNA sequencing.

Construct Expression and Purification

The constructs were expressed in the bacterial periplasm by fermentationof E. coli K12 in standard media. After fermentation the cells wereheat-treated to release the periplasm content into the media. Theconstructs released into the medium were recovered by microfiltrationwith a membrane having a 0.2 μm pore size.

Each construct, now in the permeate from the filtration step, waspurified by affinity. The permeate was loaded onto a chromatographymedium containing immobilized IgG (IgG Sepharose 6FF, GE Healthcare).The loaded product was washed with phosphate buffered saline and elutedby lowering the pH.

The elution pool was adjusted to a neutral pH (pH 8) and reduced byaddition of dithiothreitol. The sample was then loaded onto an anionexchanger. After a wash step the construct was eluted in a NaCl gradientto separate it from any contaminants. The elution pool was concentratedby ultrafiltration to 40-50 mg/ml. It should be noted that thesuccessful affinity purification of a construct on an immobilized IgGmedium indicates that the construct in question has a high affinity toIgG.

The purified ligands were analyzed with RPC LC-MS to determine thepurity and to ascertain that the molecular weight corresponded to theexpected (based on the amino acid sequence).

Example 1

The purified monomeric ligands listed in Table 1, further comprising forSEQ ID NO 8-16, 23-28 and 36-48 an AQGT leader sequence at theN-terminus and a cysteine at the C terminus, were immobilized on BiacoreCM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kitof GE Healthcare (for carbodiimide coupling of amines on thecarboxymethyl groups on the chip) in an amount sufficient to give asignal strength of about 200-1500 RU in a Biacore surface plasmonresonance (SPR) instrument (GE Healthcare, Sweden). To follow the IgGbinding capacity of the immobilized surface 1 mg/ml human polyclonal IgG(Gammanorm) was flowed over the chip and the signal strength(proportional to the amount of binding) was noted. The surface was thencleaned-in-place (CIP), i.e. flushed with 500 mM NaOH for 10 minutes atroom temperature (22+/−2° C.). This was repeated for 96-100 cycles andthe immobilized ligand alkaline stability was followed as the remainingIgG binding capacity (signal strength) after each cycle. The results areshown in Table 1 and indicate that at least the ligands Zvar(N11K)1,Zvar(N11E)1, Zvar(N11Y)1, Zvar(N11T)1, Zvar(N11F)1, Zvar(N11L)1,Zvar(N11W)1, ZN11|)1, Zvar(N11M)1, Zvar(N11V)1, Zvar(N11A)1,Zvar(N11H1), Zvar(N11R)1, Zvar(N11E,Q32A)1, Zvar(N11E,Q32E,Q40E)1 andZvar(N11E,Q32E,K50R)1, Zvar(Q9A,N11E,N43A)1, Zvar(Q9A,N11E,N28A,N43A)1,Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)1,Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1,Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)1,Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1,Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1, Zvar(N11K,H18K, D37E, A42R, N43A, L44I)1, Zvar(Q9A, N11K, H18K, D37E, A42R, N43A,L44I)1 and Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)1, as wellas the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 havingG,S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K in position 29, the varieties ofZvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 having F,Y,W,K or R in position 53and the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 where Q9, Q40,A42 or N43 has been deleted, have an improved alkali stability comparedto the parental structure Zvar1, used as the reference. Further, theligands B(Q9A,N11E,Q40V,A42K,N43A,L44I)1 and C(Q9A,N11E,E43A)1 have animproved stability compared to the parental B and C domains, used asreferences.

TABLE 1 Monomeric ligands, evaluated by Biacore (0.5M NaOH). CapacityReference Capacity after 96- capacity relative 100 after 96- to LigandSequence cycles 100 cycles reference Zvar(N11E, Q32A)1 SEQ ID 57% 55%1.036 NO 12 Zvar(N11E)1 SEQ ID 59% 55% 1.073 NO 13 Zvar(N11E, Q32E,Q40E)1 SEQ ID 52% 51% 1.020 NO 14 Zvar(N11E, Q32E, K50R)1 SEQ ID 53% 51%1.039 NO 15 Zvar(N11K)1 SEQ ID 62% 49% 1.270 NO 16 Zvar(N11Y)1 SEQ ID55% 46% 1.20 NO 38 Zvar(N11T)1 SEQ ID 50% 46% 1.09 NO 39 Zvar(N11F)1 SEQID 55% 46% 1.20 NO 40 Zvar(N11L)1 SEQ ID 57% 47% 1.21 NO 41 Zvar(N11W)1SEQ ID 57% 47% 1.21 NO 42 Zvar(N11I)1 SEQ ID 57% 47% 1.21 NO 43Zvar(N11M)1 SEQ ID 58% 46% 1.26 NO 44 Zvar(N11V)1 SEQ ID 56% 46% 1.22 NO45 Zvar(N11A)1 SEQ ID 58% 46% 1.26 NO 46 Zvar(N11H)1 SEQ ID 57% 46% 1.24NO 47 Zvar(N11R)1 SEQ ID 59% 46% 1.28 NO 48 Zvar(Q9A, N11E, N43A)1 SEQID 70% 47% 1.49 NO 8  Zvar(Q9A, N11E, N28A, N43A)1 SEQ ID 68% 47% 1.45NO 9  Zvar(Q9A, N11E, Q40V, A42K, N43E, L44I)1 SEQ ID 67% 47% 1.43 NO 10Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)1 SEQ ID 66% 47% 1.40 NO 11Zvar(Q9A, N11E, N28A, Q40V, A42K, N43A, L44I)1 SEQ ID 65% 48% 1.35 NO 24Zvar(N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y)1 SEQ ID 67%46% 1.46 NO 23 Zvar(Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R,SEQ ID 59% 46% 1.28 L51Y)1 NO 25 Zvar(N11K, H18K, D37E, A42R, N43A,L44I)1 SEQ ID 59% 45% 1.31 NO 26 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A,L44I)1 SEQ ID 63% 45% 1.40 NO 27 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A,L44I, K50R)1 SEQ ID 67% 45% 1.49 NO 28 B(Q9A, N11E, Q40V, A42K, N43A,L44I)1 SEQ ID 39% 35% 1.11 NO 36 C(Q9A, N11E, E43A)1 SEQ ID 60% 49% 1.22NO 37 Zvar(Q9A, N11E, A29G, Q40V, A42K, N43A, L44I)1 SEQ ID 69% 48% 1.44NO 54 Zvar(Q9A, N11E, A29S, Q40V, A42K, N43A, L44I)1 SEQ ID 66% 48% 1.38NO 55 Zvar(Q9A, N11E, A29Y, Q40V, A42K, N43A, L44I)1 SEQ ID 61% 48% 1.27NO 56 Zvar(Q9A, N11E, A29Q, Q40V, A42K, N43A, L44I)1 SEQ ID 60% 47% 1.28NO 57 Zvar(Q9A, N11E, A29T, Q40V, A42K, N43A, L44I)1 SEQ ID 60% 47% 1.28NO 58 Zvar(Q9A, N11E, A29N, Q40V, A42K, N43A, L44I)1 SEQ ID 61% 47% 1.30NO 59 Zvar(Q9A, N11E, A29F, Q40V, A42K, N43A, L44I)1 SEQ ID 62% 46% 1.35NO 60 Zvar(Q9A, N11E, A29L, Q40V, A42K, N43A, L44I)1 SEQ ID 61% 46% 1.33NO 61 Zvar(Q9A, N11E, A29W, Q40V, A42K, N43A, L44I)1 SEQ ID 60% 46% 1.30NO 62 Zvar(Q9A, N11E, A29I, Q40V, A42K, N43A, L44I)1 SEQ ID 58% 47% 1.23NO 63 Zvar(Q9A, N11E, A29M, Q40V, A42K, N43A, L44I)1 SEQ ID 62% 47% 1.32NO 64 Zvar(Q9A, N11E, A29V, Q40V, A42K, N43A, L44I)1 SEQ ID 62% 47% 1.32NO 65 Zvar(Q9A, N11E, A29D, Q40V, A42K, N43A, L44I)1 SEQ ID 56% 47% 1.19NO 66 Zvar(Q9A, N11E, A29E, Q40V, A42K, N43A, L44I)1 SEQ ID 57% 47% 1.21NO 67 Zvar(Q9A, N11E, A29H, Q40V, A42K, N43A, L44I)1 SEQ ID 57% 47% 1.21NO 68 Zvar(Q9A, N11E, A29R, Q40V, A42K, N43A, L44I)1 SEQ ID 58% 46% 1.26NO 69 Zvar(Q9A, N11E, A29K, Q40V, A42K, N43A, L44I)1 SEQ ID 59% 46% 1.28NO 70 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53F)1 SEQ ID 58% 46% 1.26NO 71 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53Y)1 SEQ ID 59% 46% 1.28NO 72 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53W)1 SEQ ID 62% 46% 1.35NO 73 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53K)1 SEQ ID 65% 46% 1.41NO 74 Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I, D53R)1 SEQ ID 60% 46% 1.30NO 75 Zvar(Q9del, N11E, Q40V, A42K, N43A, L44I)1 SEQ ID 60% 46% 1.30 NO76 Zvar(Q9A, N11E, Q40del, A42K, N43A, L44I)1 SEQ ID 59% 46% 1.28 NO 77Zvar(Q9A, N11E, Q40V, A42del, N43A, L44I)1 SEQ ID 57% 46% 1.24 NO 78Zvar(Q9A, N11E, Q40V, A42K, N43del, L44I)1 SEQ ID 55% 46% 1.20 NO 79

The Biacore experiment can also be used to determine the binding anddissociation rates between the ligand and IgG. This was used with theset-up as described above and with an IgG1 monoclonal antibody as probemolecule. For the reference Zvar1, the on-rate (105 M⁻¹s⁻¹) was 3.1 andthe off-rate (10⁵ s⁻¹) was 22.1, giving an affinity (off-rate/on-rate)of 713 pM. For Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 (SEQ ID NO. 11), theon-rate was 4.1 and the off-rate 43.7, with affinity 1070 pM. The IgGaffinity was thus somewhat higher for the mutated variant.

Example 2

The purified dimeric, tetrameric and hexameric ligands listed in Table 2were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden),using the amine coupling kit of GE Healthcare (for carbodiimide couplingof amines on the carboxymethyl groups on the chip) in an amountsufficient to give a signal strength of about 200-1500 RU in a Biacoreinstrument (GE Healthcare, Sweden). To follow the IgG binding capacityof the immobilized surface 1 mg/ml human polyclonal IgG (Gammanorm) wasflowed over the chip and the signal strength (proportional to the amountof binding) was noted. The surface was then cleaned-in-place (CIP), i.e.flushed with 500 mM NaOH for 10 minutes at room temperature (22+/−2°C.). This was repeated for 300 cycles and the immobilized ligandalkaline stability was followed as the remaining IgG binding capacity(signal strength) after each cycle. The results are shown in Table 2 andin FIG. 2 and indicate that at least the ligands Zvar(Q9A,N11E,N43A)4,Zvar(Q9A,N11E,N28A,N43A)4, Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4 andZvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4,Zvar(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)4 andZvar(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)4 have an improved alkalistability compared to the parental structure Zvar4, which was used as areference. The hexameric ligand Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)6 alsohas improved alkali stability compared to the parental structure Zvar6,used as a reference. Further, Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I) dimerswith deletions of a) D2,A3,K4; b) K58,V1,D2; c) P57,K58,V1,D2,A3; d)K4,F5,D6,K7,E8; e) A56,P57,K58; V1,D2,A3 or f) V1,D2,A3,K4,F5,D6,K7,E8from the linker region between the two monomer units have improvedalkali stability compared to the parental structure Zvar2, used as areference. Also Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I) dimers with aninsertion of YEDG between K58 and V1 in the linker region have improvedalkali stability compared to Zvar2.

TABLE 2 Dimeric, tetrameric and hexameric ligands, evaluated by Biacore(0.5M NaOH). Capacity Capacity Capacity Remaining relative Remainingrelative Remaining relative SEQ capacity 100 to ref. capacity 200 toref. capacity 300 to ref. Ligand ID NO: cycles (%) 100 cycles cycles (%)200 cycles cycles (%) 300 cycles Zvar4 21 67 1 36 1 16 1 Zvar(Q9A, N11E,N43A)4 17 81 1.21 62 1.72 41 2.56 Zvar(Q9A, N11E, N28A, 18 80 1.19 621.72 42 2.62 N43A)4 Zvar(Q9A, N11E, Q40V, 19 84 1.25 65 1.81 48 3.00A42K, N43E, L44I)4 Zvar(Q9A, N11E, Q40V, 20 90 1.34 74 2.06 57 3.56A42K, N43A, L44I)4 Zvar(Q9A, N11E, N28A, 32 84 1.24 Not tested Nottested Not tested Not tested Q40V, A42K, N43A, L44I)4 Zvar(Q9A, N11E,Q40V, 33 87 1.30 Not tested Not tested Not tested Not tested A42K, N43A,L44I)6 Zvar(Q9A, N11E, D37E, 34 81 1.13 Not tested Not tested Not testedNot tested Q40V, A42K, N43A, L44I)4 Zvar(Q9A, N1E, D37E, 35 84 1.17 Nottested Not tested Not tested Not tested Q40V, A42R, N43A, L44I)4Zvar(Q9A, N11E, Q40V, 80 70 1.27 Not tested Not tested Not tested Nottested A42K, N43A, L44I)2 with D2, A3 and K4 in linker deleted Zvar(Q9A,N11E, Q40V, 81 76 1.38 Not tested Not tested Not tested Not tested A42K,N43A, L44I)2 with K58, V1 and D2 in linker deleted Zvar(Q9A, N11E, Q40V,82 74 1.35 Not tested Not tested Not tested Not tested A42K, N43A,L44I)2 with P57, K58, V1, D2 and A3 in linker deleted Zvar(Q9A, N11E,Q40V, 83 70 1.30 Not tested Not tested Not tested Not tested A42K, N43A,L44I)2 with K4, F5, D6, K7 and E8 in linker deleted Zvar(Q9A, N11E,Q40V, 84 68 1.26 Not tested Not tested Not tested Not tested A42K, N43A,L44I)2 with A56, P57 and K58 in linker deleted Zvar(Q9A, N11E, Q40V, 8575 1.39 Not tested Not tested Not tested Not tested A42K, N43A, L44I)2with V1, D2 and A3 in linker deleted Zvar(Q9A, N11E, Q40V, 86 62 1.13Not tested Not tested Not tested Not tested A42K, N43A, L44I)2 with V1,D2, A3, K4, F5, D6, K7 and E8 in linker deleted Zvar(Q9A, N11E, Q40V, 8772 1.31 Not tested Not tested Not tested Not tested A42K, N43A, L44I)2with YEDG inserted in linker between K58 and V1 Zvar2 88 55 1 Not testedNot tested Not tested Not tested

Example 3

Example 2 was repeated with 100 CIP cycles of three ligands using 1 MNaOH instead of 500 mM as in Example 2. The results are shown in Table 3and show that all three ligands have an improved alkali stability alsoin 1M NaOH, compared to the parental structure Zvar4 which was used as areference.

TABLE 3 Tetrameric ligands, evaluated by Biacore (1M NaOH). RemainingCapacity capacity 100 relative to Ligand Sequence cycles (%) ref. 100cycles Zvar4 SEQ ID NO 21 27 1 Zvar(Q9A, N11E, N28A, N43A)4 SEQ ID NO 1855 2.04 Zvar(Q9A, N11E, Q40V, A42K, N43E, L44I)4 SEQ ID NO 19 54 2.00Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)4 SEQ ID NO 20 56 2.07

Example 4

The purified tetrameric ligands of Table 2 (all with an additionalN-terminal cysteine) were immobilized on agarose beads using the methodsdescribed below and assessed for capacity and stability. The results areshown in Table 4 and FIG. 3.

TABLE 4 Matrices with tetrametric ligands, evaluated in columns (0.5MNaOH). Remaining Remaining Capacity IgG capacity IgG capacity retentionLigand Initial IgG Qb10 after after six 4 h relative to SEQ contentcapacity Qb10 six 4 h cycles cycles ref. after six Ligand ID NO. (mg/ml)(mg/ml) (mg/ml) (%) 4 h cycles Zvar4 21 7 52.5 36.5 60 1 Zvar4 21 1261.1 43.4 71 1 Zvar(Q9A, N11E, N28A, 18 7.0 49.1 44.1 90 1.50 N43A)4Zvar(Q9A, N11E, N28A, 18 12.1 50.0 46.2 93 1.31 N43A)4 Zvar(Q9A, N11E,Q40V, 20 7.2 49.0 44.2 90 1.50 A42K, N43A, L44I)4 Zvar(Q9A, N11E, Q40V,20 12.8 56.3 53.6 95 1.34 A42K, N43A, L44I)4 Zvar(N11K, H18K, S33K, 309.7 56.3 52.0 92 1.53 D37E, A42R, N43A, L44I, K50R, L51Y)4 Zvar(Q9A,N11K, H18K, 31 10.8 56.9 52.5 92 1.30 D37E, A42R)4

Activation

The base matrix used was rigid cross-linked agarose beads of 85micrometers (volume-weighted, d50V) median diameter, prepared accordingto the methods of U.S. Pat. No. 6,602,990, hereby incorporated byreference in its entirety, and with a pore size corresponding to aninverse gel filtration chromatography Kay value of 0.70 for dextran ofMw 110 kDa, according to the methods described in Gel FiltrationPrinciples and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13. 25 mL(g) of drained base matrix, 10.0 mL distilled water and 2.02 g NaOH (s)was mixed in a 100 mL flask with mechanical stirring for 10 min at 25°C. 4.0 mL of epichlorohydrin was added and the reaction progressed for 2hours. The activated gel was washed with 10 gel sediment volumes (GV) ofwater.

Coupling

To 20 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 169 mgNaHCO₃, 21 mg Na₂CO₃, 175 mg NaCl and 7 mg EDTA, was added. The Falcontube was placed on a roller table for 5-10 min, and then 77 mg of DTEwas added. Reduction proceeded for >45 min. The ligand solution was thendesalted on a PD10 column packed with Sephadex G-25. The ligand contentin the desalted solution was determined by measuring the 276 nm UVabsorption.

The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH8.6} and the ligand was then coupled according to the method describedin U.S. Pat. No. 6,399,750, hereby incorporated by reference in itsentirety. All buffers used in the experiments had been degassed bynitrogen gas for at least 5-10 min. The ligand content of the gels couldbe controlled by varying the amount and concentration of the ligandsolution.

After immobilization the gels were washed 3×GV with distilled water. Thegels+1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixedand the tubes were left in a shaking table at room temperatureovernight. The gels were then washed alternately with 3×GV {0.1 MTRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10×GV with distilledwater. Gel samples were sent to an external laboratory for amino acidanalysis and the ligand content (mg/ml gel) was calculated from thetotal amino acid content.

Protein

Gammanorm 165 mg/ml (Octapharma), diluted to 2 mg/ml in Equilibrationbuffer.

Equilibration Buffer

PBS Phosphate buffer 10 mM+0.14 M NaCl+0.0027 M KCl, pH 7,4 (Medicago)

Adsorption Buffer

PBS Phosphate buffer 10 mM+0.14 M NaCl+0.0027 M KCl, pH 7,4 (Medicago)

Elution Buffers

100 mM acetate pH 2.9

Dynamic Binding Capacity

2 ml of resin was packed in TRICORN™ 5 100 columns. The breakthroughcapacity was determined with an ÅKTAExplorer 10 system at a residencetime of 6 minutes (0.33 ml/min flow rate). Equilibration buffer was runthrough the bypass column until a stable baseline was obtained. This wasdone prior to auto zeroing. Sample was applied to the column until a100% UV signal was obtained. Then, equilibration buffer was appliedagain until a stable baseline was obtained.

Sample was loaded onto the column until a UV signal of 85% of maximumabsorbance was reached. The column was then washed with 5 column volumes(CV) equilibration buffer at flow rate 0.5 ml/min. The protein waseluted with 5 CV elution buffer at a flow rate of 0.5 ml/min. Then thecolumn was cleaned with 0.5M NaOH at flow rate 0.2 ml/min andre-equilibrated with equilibration buffer.

For calculation of breakthrough capacity at 10%, the equation below wasused. That is the amount of IgG that is loaded onto the column until theconcentration of IgG in the column effluent is 10% of the IgGconcentration in the feed.

${q_{10\%} = {\frac{c_{0}}{V_{c}}\left\lbrack {V_{app} - V_{sys} - {\int_{V_{sys}}^{V_{app}}{\frac{{A(V)} - A_{sub}}{A_{100\%} - A_{sub}}*{dv}}}} \right\rbrack}}\ $

-   -   A_(100%)=100% UV signal;    -   A_(sub)=absorbance contribution from non-binding IgG subclass;    -   A(V)=absorbance at a given applied volume;    -   V_(c)=column volume;    -   V_(app)=volume applied until 10% breakthrough;    -   V_(sys)=system dead volume;    -   C₀=feed concentration.

The dynamic binding capacity (DBC) at 10% breakthrough was calculated.The dynamic binding capacity (DBC) was calculated for 10 and 80%breakthrough.

CIP—0.5 M NaOH

The 10% breakthrough DBC (Qb10) was determined both before and afterrepeated exposures to alkaline cleaning solutions. Each cycle included aCIP step with 0.5 M NaOH pumped through the column at a rate of 0.5/minfor 20 min, after which the column was left standing for 4 h. Theexposure took place at room temperature (22+/−2° C.). After thisincubation, the column was washed with equilibration buffer for 20 minat a flow rate of 0.5 ml/min. Table 4 shows the remaining capacity aftersix 4 h cycles (i.e. 24 h cumulative exposure time to 0.5 M NaOH), bothin absolute numbers and relative to the initial capacity.

Example 5

Example 4 was repeated with the tetrameric ligands shown in Table 5, butwith 1.0 M NaOH used in the CIP steps instead of 0.5 M. The results areshown in Table 5 and in FIG. 4.

TABLE 5 Matrices with tetrametric ligands, evaluated in columns - 1.0MNaOH. Remaining Remaining Capacity IgG capacity IgG capacity retentionLigand Initial IgG Qb10 after after six 4 h relative to SEQ contentcapacity Qb10 six 4 h cycles cycles ref. after six Ligand ID NO. (mg/ml)(mg/ml) (mg/ml) (%) 4 h cycles Zvar4 21 12 60.1 33.5 56 1 Zvar(Q9A,N11E, Q40V, 20 12.8 60.3 56.0 93 1.67 A42K, N43A, L44I)4 Zvar(N11K,H18K, S33K, 30 9.7 62.1 48.1 77 1.44 D37E, A42R, N43A, L44I, K50R,L51Y)4

Example 6 Base Matrices

The base matrices used were a set of rigid cross-linked agarose beadsamples of 59-93 micrometers (volume-weighted, d50V) median diameter(determined on a Malvern Mastersizer 2000 laser diffraction instrument),prepared according to the methods of U.S. Pat. No. 6,602,990 and with apore size corresponding to an inverse gel filtration chromatography Kdvalue of 0.62-0.82 for dextran of Mw 110 kDa, according to the methodsdescribed above, using HR10/30 columns (GE Healthcare) packed with theprototypes in 0.2 M NaCl and with a range of dextran fractions as probemolecules (flow rate 0.2 ml/min). The dry weight of the bead samplesranged from 53 to 86 mg/ml, as determined by drying 1.0 ml drainedfilter cake samples at 105° C. overnight and weighing.

TABLE 6 Base matrix samples Base matrix Kd d50v (μm) Dry weight (mg/ml)A18 0.704 59.0 56.0 A20 0.70 69.2 55.8 A27 0.633 87.2 74.2 A28 0.63867.4 70.2 A29 0.655 92.6 57.5 A32 0.654 73.0 70.5 A33 0.760 73.1 55.5A38 0.657 70.9 56.2 A39 0.654 66.0 79.1 A40 0.687 64.9 74.9 A41 0.70881.7 67.0 A42 0.638 88.0 59.4 A43 0.689 87.5 77.0 A45 0.670 56.6 66.0A52 0.620 53.10 63.70 A53 0.630 52.6 86.0 A54 0.670 61.3 75.3 A55 0.64062.0 69.6 A56 0.740 61.0 56.0 A56-2 0.740 51.0 56.0 A62a 0.788 48.8 70.1A62b 0.823 50.0 46.9 A63a 0.790 66.8 59.6 A63b 0.765 54.0 79.0 A65a0.796 58.0 60.0 A65b 0.805 57.3 46.0 B5 0.793 69.0 84.4 C1 0.699 71.073.4 C2 0.642 66.5 81.1 C3 0.711 62.0 82.0 C4 0.760 62.0 82.0 H31 0.71782.0 59.0 H35 0.710 81.1 61.0 H40 0.650 52.8 65.0 I1 0.640 50.0 67.0 410.702 81.6 60.6

Coupling

100 ml base matrix was washed with 10 gel volumes distilled water on aglass filter. The gel was weighed (1 g=1 ml) and mixed with 30 mldistilled water and 8.08 g NaOH (0.202 mol) in a 250 ml flask with anagitator. The temperature was adjusted to 27+/−2° C. in a water bath. 16ml epichlorohydrin (0.202 mol) was added under vigorous agitation (about250 rpm) during 90+/−10 minutes. The reaction was allowed to continuefor another 80+/−10 minutes and the gel was then washed with >10 gelvolumes distilled water on a glass filter until neutral pH was reached.This activated gel was used directly for coupling as below.

To 16.4 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 139 mgNaHCO₃, 17.4 mg Na₂CO₃, 143.8 mg NaCl and 141 mg EDTA, was added. TheFalcon tube was placed on a roller table for 5-10 min, and then 63 mg ofDTE was added. Reduction proceeded for >45 min. The ligand solution wasthen desalted on a PD10 column packed with Sephadex G-25. The ligandcontent in the desalted solution was determined by measuring the 276 nmUV absorption.

The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH8.6} and the ligand was then coupled according to the method describedin U.S. Pat. No. 6,399,750 5.2.2, although with considerably higherligand amounts (see below). All buffers used in the experiments had beendegassed by nitrogen gas for at least 5-10 min. The ligand content ofthe gels was controlled by varying the amount and concentration of theligand solution, adding 5-20 mg ligand per ml gel. The ligand was eithera tetramer (SEQ ID NO. 20) or a hexamer (SEQ ID NO. 33) of analkali-stabilized mutant.

After immobilization the gels were washed 3×GV with distilled water. Thegels+1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixedand the tubes were left in a shaking table at room temperatureovernight. The gels were then washed alternately with 3×GV {0.1 MTRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10×GV with distilledwater. Gel samples were sent to an external laboratory for amino acidanalysis and the ligand content (mg/ml gel) was calculated from thetotal amino acid content.

Evaluation

The Qb10% dynamic capacity for polyclonal human IgG at 2.4 and 6 minresidence time was determined as outlined in Example 4.

TABLE 7 Prototype results Ligand Qb10% Qb10% Base content 2.4 min 6 minPrototype matrix (mg/ml) Multimer (mg/ml) (mg/ml) N1 A38 7.45 tetramer44.4 58.25 N2 A20 7.3 tetramer 45.12 57.21 N3 A42 6.72 tetramer 33.5650.02 N4 A29 7.3 tetramer 36.34 51.8 N5 A28 7.9 tetramer 42.38 58.25 N6A39 6.96 tetramer 41.88 54.67 N7 A27 7.5 tetramer 29.19 48.73 N8 A436.99 tetramer 33.43 49.79 N9 A38 11.34 tetramer 48.1 72.78 N10 A20 10.6tetramer 50.66 70.07 N11 A42 11.1 tetramer 32.25 57.78 N12 A29 11tetramer 34.85 64.68 N13 A28 11.9 tetramer 39.92 63.75 N14 A39 10.48tetramer 44.37 64.79 N15 A27 12.1 tetramer 24.8 55.56 N16 A43 10.51tetramer 31.82 58.04 N17 A41 8.83 tetramer 38.5 56.8 N18 A41 8.83tetramer 37.84 58.6 N19 A41 8.83 tetramer 35.06 57.23 N20 A41 5.0tetramer 35.64 46.04 N21 A41 13.0 tetramer 34.95 62.23 N22 A40 13.15tetramer 56.85 71.09 N23 A33 7.33 tetramer 48.69 55.76 N24 A40 11.03tetramer 54.96 73.8 033A A38 7.5 tetramer 44 58 033B A38 11.3 tetramer48 73 097A A20 7.3 tetramer 45 57 097B A20 10.6 tetramer 51 70 003A A287.9 tetramer 42 58 003B A28 11.9 tetramer 40 64 003C A28 15.8 tetramer37 67 038A A39 7.0 tetramer 42 55 038B A39 10.5 tetramer 44 65 074 A4013.2 tetramer 57 71 093 A33 7.3 tetramer 49 56 058A A40 11.0 tetramer 5574 077 A18 8.2 tetramer 52 59 010 A32 10.7 tetramer 40 57 099 A32 13.3tetramer 37 66 030A B5 6.3 tetramer 32 38 030B B5 9.6 tetramer 45 47293A C1 5.4 tetramer 38 47 293B C1 10.8 tetramer 43 60 294A C2 5.1tetramer 39 46 294B C2 10.5 tetramer 42 57 336A H40 5.6 tetramer 47 52336B H40 9.1 tetramer 52 67 091 A18 13.4 tetramer N/A 63 092 A20 12.8tetramer 49 67 080 A33 9.4 tetramer 51 58 089 A40 6.1 tetramer 49 59688A A62a 6.6 tetramer 41 46 688B A62a 14.8 tetramer 55 62 871 A62a 9.7tetramer 48 60 934A A63a 6.6 tetramer 40 44 934B A63a 14.0 tetramer 4856 017B A65a 13.1 tetramer 56 64 041A A62b 5.2 tetramer 40 N/A 041B A62b11.1 tetramer 52 N/A 116A A65b 5.8 tetramer 42 46 116B A65b 8.8 tetramer49 56 017A A65a 6.1 tetramer 40 44 387A A62a 6.4 tetramer 43 45 387BA62a 7.5 tetramer 47 56 432 A63a 6.1 tetramer 39 44 433A A65a 6.6tetramer 42 47 433B A65a 13.6 tetramer 52 61 579A I1 6.1 tetramer 45 51579B I1 11.2 tetramer 57 68 064A C3 5.9 tetramer 44 52 064B C3 9.0tetramer 49 62 064C C3 14.3 tetramer 51 70 352A C4 10.1 tetramer 55 63352B C4 14.4 tetramer 59 67 066A C3 6.8 hexamer 48 59 066B C3 11.9hexamer 51 73 066C C3 15.1 hexamer 43 61 353A C4 11.2 hexamer 62 74 353BC4 15.2 hexamer 57 82 872A A62a 9.6 hexamer 56 72 872B A62a 14.5 hexamer62 84 869A H40 6.9 hexamer 50 56 869B H40 14.3 hexamer 56 75 869C H4023.0 hexamer 41 65 962A H35 6.8 hexamer 36 49 962B H35 12.3 hexamer 3154 962C H35 20.3 hexamer 20 43 112A A56 7.9 hexamer 47 55 112B A56 12.4hexamer 57 73 112C A56 19.2 hexamer 55 80 113A A56 7.1 hexamer 48 57113B A56 12.4 hexamer 53 73 113C A56 15.2 hexamer 48 76 212A H31 6.5hexamer 37 38 212B H31 10.4 hexamer 50 61 212C H31 20.0 hexamer 31 52213A A33 6.5 hexamer 44 53 213B A33 10.9 hexamer 50 65 213C A33 11.1hexamer 50 68 432A A20 6.4 hexamer 41 56 432B A20 12.4 hexamer 38 64432C A20 21.1 hexamer 44 43 433A A38 5.9 hexamer 47 57 433B A38 11.6hexamer 48 72 433C A38 15.8 hexamer 36 62 742A A54 6.7 hexamer 38 46742B A54 12.6 hexamer 45 52 742C A54 21.1 hexamer 38 65 726A A63b 6.4hexamer 42 46 726B A63b 10.6 hexamer 49 60 726C A63b 16.7 hexamer 53 69793A A56-2 6.8 hexamer 50 58 793B A56-2 12.5 hexamer 59 72 793C A56-219.2 hexamer 61 82

Example 7

A series of prototypes, prepared as above, with different ligand content(tetramer, SEQ ID NO:20) were incubated in 1 M NaOH for 4, 8 and 31hours at 22+/−2° C. and the dynamic IgG capacity (Qb10%, 6 min residencetime) was measured before and after incubation. The prototypes are shownin Table 8 and the results in FIGS. 5 and 6. It can be seen that thestability towards this harsh alkali treatment increases with increasingligand content.

TABLE 8 Samples for incubation in 1M NaOH Qb10%, 6 min, before PrototypeLigand content (mg/ml) incubation (mg/ml) N1 12 78 LE28 13 79 N17 16 73N16 20 73

Example 8

Two crosslinked agarose bead prototypes, prepared as above, withdifferent ligand content (hexamer, SEQ ID NO:33), median bead diameter(d50,v) 62 μm and Kd 0.70 for dextran of Mw 110 kD, were evaluated witha real mAb feed. The ligand content of prototype A was 14.3 mg/ml and ofprototype B 18.9 mg/ml. For comparison, the commercial product MabSelectSuRe® LX (GE Healthcare Life Sciences, with ligand SEQ ID NO. 21) wasused. The resins were packed in Tricorn columns (GE Healthcare LifeSciences) to bed heights of 10 cm, giving bed volumes of 2 ml and thecolumns were shown to have peak asymmetry within the 0.8-1.5 interval.The sample loaded was a clarified CHO cell supernatant with 4.9 mg/mlmonoclonal IgG1 antibody at physiological pH and the experimentalconditions were as listed below in Table 9 (CV=column volumes,RT=residence time).

TABLE 9 Conditions for evaluation with real feed. Equilibration: 3 CV 20mM phosphate, 150 mM NaCl pH 7.4, RT = 3.4 min Sample loading: 43 mgmAb/ml resin, RT = 6 min Wash 1: 5 CV 20 mM phosphate, 500 mM NaCl pH7.4, 1.5 CV at RT = 6 min and 3.5 CV at RT = 3.4 min Wash 2: 1 CV 50 mMacetate pH 6.0, RT = 3.4 min Elution: 3 CV 50 mM acetate pH 3.5, RT = 6min, peak collected between 150 mAU-150 mAU Strip: 2 CV 100 mM acetate,RT = 3.4 min CIP: 3 CV 0.1M NaOH, RT = 6 min Re-equilibration: 5 CV 20mM phosphate, 150 mM NaCl pH 7.4, RT = 3.4 min

The mAb peak was collected using a UV watch function and theconcentration of the mAb was determined by UV measurement at 280 nm(extinction coefficient 1.5). All absorbance detections were performedusing a spectrophotometer, including the measurements for the yieldcalculations.

Samples for HCP (host cell protein) analyses were prepared by adding 10%Preservation buffer (0.2 M NaH₂PO₄*H₂O (5.3%), 0.2 M Na₂HPO₄*12 H₂O(94.7%), 0.5% Tween 20, 1% BSA pH 8) to the samples directly after eachrun made (e.g. 50 μl preservation buffer to 450 μl sample). The HCPcontent was measured using commercial anti-CHO antibodies (CygnusTechnologies) and a Gyrolab (Gyros AB, Sweden) work station.

The results are presented in Table 10 below and show that theperformance of the prototypes is in the same range as for the commercialproduct. The HCP content in the feed was 331 000 ppm.

TABLE 10 Results from real feed evaluation Resin Yield (%) Elution pool(CV) HCP in pool (ppm) MabSelect SuRe LX 90 1.5 914 MabSelect SuRe LX 951.6 1021 Prototype A 96 1.3 1076 Prototype A 95 1.3 1105 Prototype B 961.3 1040 Prototype B 93 1.3 1104

Example 9

A crosslinked agarose bead matrix prototype, prepared as above, with14.5 mg/ml ligand (hexamer, SEQ ID NO:33), median bead diameter (d50,v)57.4 μm, Kd 0.72 for dextran of Mw 110 kD and dry weight 70.3 mg/ml, wasevaluated for elution pH with two real mAb feeds (mAb1 2.4 g/I and mAb24.9 g/l) IgG1, physiological pH, and a sample of polyclonal human IgG(Gammanorm, Octapharma). For comparison, the commercial productMabSelect SuRe® LX (GE Healthcare Life Sciences) was used. The resinswere packed in Tricorn columns (GE Healthcare Life Sciences) to bedheights of 10 cm, giving bed volumes of 2 ml and the columns were shownto have peak asymmetry within the 0.8-1.5 interval. The samples loadedwere clarified CHO cell supernatants with IgG1 mAbs at physiological pHand the experimental conditions were as listed below in Table 11(CV=column volumes, RT=residence time).

TABLE 11 Conditions for elution pH evaluation. Equilibration: 5 CV 20 mMphosphate, 150 mM NaCl pH 7.4, RT = 3.4 min Sample loading: 10 mg mAb/mlresin, RT = 6 min Wash: 6 CV 20 mM phosphate, 150 mM NaCl pH 7.4, RT =3.4 min Elution: 30 CV 100 mM citrate pH 6-3 gradient, RT = 6 min CIP: 3CV 0.1M NaOH, RT = 6 min Re-equilibration: 8 CV 20 mM phosphate, 150 mMNaCl pH 7.4, RT = 3.4 min

The results are shown below in Table 12 and indicate that the antibodieselute at similar pH levels as on the reference, although with someindividual variation depending on the particular antibody-resincombination.

TABLE 12 Results from elution pH evaluation Sample Elution pH MabSelectSuRe LX Elution pH prototype mAb 1 3.67 3.53 mAb 2 3.68 3.80 PolyclonalIgG 4.01 (peak 1) 4.24 (peak 1) 3.70 (peak 2) 3.81 (peak 2)

Fractions from the pH-gradient elution of polyclonal IgG were alsoanalysed with respect to content of IgG1, IgG2 and IgG4, using a BiacoreSPR instrument (GE Healthcare Life Sciences) with antibodies against thefour different IgG classes immobilized on a CM5 Biacore chip.

The chromatograms for polyclonal IgG on the reference and the prototypeare shown in FIG. 7 and the IgG class analyses are shown in FIG. 8. Thedata show that all three classes bind to both resins in a similar wayand that the first peak predominantly contains IgG2, while IgG1 and IgG4elute mainly in the second peak. The anti-IgG3 antibodies cross-reactedwith IgG4, so no reliable results for IgG3 were obtained. IgG3 isgenerally known to show no or only weak binding to Protein A.

Example 10

A crosslinked agarose bead matrix prototype, prepared as above, with12.6 mg/ml ligand (tetramer, SEQ ID NO:20), 84.9 μm median bead diameter(d50,v), Kd 0.71 for dextran Mw 110 kD and 62.2 mg/ml dry weight, wasevaluated with respect to alkali stability, using the commercial productMabSelect SuRe LX as a reference. Tricorn 5 columns packed with theresins to 10 cm bed height were flushed with 3 column volumes of 1 MNaOH. The flow was then stopped for 240 minutes (corresponding to 16normal CIP cycles of 15 min/cycle) before washing out the NaOH solutionby 3 column volumes of PBS buffer. The dynamic binding capacity forpolyclonal IgG (Gammanorm, Octapharma) was then measured and the processwas repeated with another injection of 1 M NaOH. The dynamic capacitywas measured after each 240 min incubation cycle with 1 M NaOH. In thecapacity measurements, the columns were equilibrated with PBS bufferbefore the 2 mg/ml sample was loaded (residence time 6 min) until a UVsignal of 85% of maximum absorbance was reached. Then the column waswashed with PBS buffer, eluted with 500 mM acetic acid pH 3.0 andre-equilibrated. The dynamic binding capacity at 10% and 80%breakthrough was calculated as described above. The results are shown inFIG. 9 and they show that the prototype was significantly more stablethan the commercial product.

Example 11

The long-term storage stability of a separation matrix according to theinvention was assessed. The inventive example (Inv. Ex.) was compared tothe commercial product MabSelect SuRe (MSS) (GE Healthcare LifeSciences, with ligand SEQ ID NO. 21) as a reference. The separationmatrix (Inv. Ex. or MSS) was incubated together with the storage liquidfor a predetermined period (two weeks). The storage liquids tested were20% ethanol solution; 0.01 M NaOH solution; 0.03 M NaOH solution and0.05 M NaOH solution. After incubation with the storage liquid for thepredefined period, the 10% breakthrough dynamic binding capacity at wasdetermined using human polyclonal IgG as described in Example 4, using aresidence time of 2.4 minutes. The 10% breakthrough capacity of theseparation matrices prior to storage were also determined forcomparison. The results are shown in FIG. 10 and Table 13 below.

TABLE 10 Results from long term storage evaluation Inventive ExampleMabSelect SuRe start 2 weeks 4 weeks start 2 weeks 4 weeks 20% EtOH 50.249.3 48.1 35.0 37.1 37.7 0.01M NaOH 49.7 46.2 46.0 33.2 35.2 31.3 0.03MNaOH 52.2 47.6 49.1 33.5 30.5 24.2 0.05M NaOH 51.2 48.0 49.4 34.4 26.817.8

It can be seen that the inventive example has a significantly higherinitial dynamic binding capacity as compared to the commercial MabSelectSuRe. After four weeks storage in 50 mM NaOH solution MabSelect SuReretains only approximately 52% of its original dynamic binding capacity,whereas the inventive example retains approximately 96% of its originaldynamic binding capacity. Thus, it can be seen that the separationmatrix of the inventive example is suitable for long-term storage inNaOH solution with concentrations up to at least 50 mM.

1. A method of storing a separation matrix comprising multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains covalentlycoupled to a porous support, wherein the alkali-stabilized Protein Adomains comprise mutants of a parental Fc-binding domain ofStaphylococcus Protein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO51 or 52 are asparagines and wherein at least the asparagine residue atposition 3 of SEQ ID NO 51 or 52 has been mutated to an amino acidselected from the group consisting of glutamic acid, lysine, tyrosine,threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine,valine, alanine, histidine and arginine, such as to glutamic acid; andwherein the method comprises the steps of: a) providing a storage liquidcomprising at least 50% by volume of an aqueous alkali metal hydroxidesolution; b) permeating the separation matrix with the storage liquid;and c) storing the storage liquid-permeated separation matrix for astorage time of at least 5 days.
 2. The method of claim 1, wherein themutants comprise further mutations in one or more of positions 1, 2, 7,10, 15, 20, 21, 24, 25, 28, 29, 32, 34, 35, 36, 39, 42 and 43 in SEQ IDNO 51 or
 52. 3. The method according to claim 1, wherein the glutamineresidue at position 1 of SEQ ID NO 51 or 52 has been mutated to analanine.
 4. The method according to claim 1, wherein the asparagine orglutamic acid residue at position 35 of SEQ ID NO 51 or 52 has beenmutated to an alanine.
 5. The method according to claim 1, wherein themultimers of immunoglobulin-binding alkali-stabilized Protein A domainsare homomultimers selected from the group consisting of dimers, trimers,tetramers, pentamers, hexamers, heptamers, octamers or nonamers.
 6. Themethod according to claim 1, wherein the multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains each comprisea C-terminal cysteine residue for covalent coupling to the poroussupport.
 7. The method according to claim 1, wherein the multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains are coupledto the porous support via thioether links.
 8. The method according toclaim 1, wherein the separation matrix comprises at least 11 mg/ml, suchas at least 15 mg/ml, of the multimers of immunoglobulin-bindingalkali-stabilized Protein A domains covalently coupled to the poroussupport.
 9. The method according to claim 1, wherein the porous supportis highly cross-linked agarose beads.
 10. The method according to claim1, wherein the aqueous alkali metal hydroxide solution is sodiumhydroxide solution, potassium hydroxide solution or a mixture thereof,preferably sodium hydroxide solution.
 11. The method according to claim1, wherein the aqueous alkali metal hydroxide solution has a molarity offrom 10 mM to 100 mM, such as from 30 mM to 50 mM.
 12. The methodaccording to claim 1, wherein the storage liquid further comprises aC2-C7 alcohol, such as ethanol, isopropanol or benzyl alcohol.
 13. Themethod according to claim 1, wherein the storage liquid comprises atleast 70% by volume aqueous alkali metal hydroxide solution, such as atleast 90% by volume aqueous alkali metal hydroxide solution, preferablyat least 99% by volume aqueous alkali metal hydroxide solution.
 14. Themethod according to claim 1, wherein the storage time is at least 10days, such as at least 25 days, or such as at least 50 days, or such asat least 100 days, or such as at least 200 days, such as up to 400 days,or such as up to 700 days.
 15. The method according to claim 1, whereinthe separation matrix is cleaned and/or sanitized with a cleaning fluidprior to storing, wherein the cleaning fluid comprises at least 50% byvolume of an aqueous alkali metal hydroxide solution, and wherein theaqueous alkali metal hydroxide solution has a molarity of from 500 mM to5 M, such as from 1 M to 2 M.
 16. The method according to claim 1,wherein the separation matrix retains at least 80% of its originaldynamic binding capacity after step b), such as at least 90% of itsoriginal dynamic binding capacity.
 17. Use of a storage liquidcomprising at least 50% by volume of an aqueous alkali metal hydroxidesolution for the storage of a separation matrix comprising multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains covalentlycoupled to a porous support.
 18. A separation matrix product comprisinga storage receptacle, a separation matrix and a storage liquid; whereinthe storage receptacle contains the separation matrix permeated with thestorage liquid; wherein the separation matrix comprises multimers ofimmunoglobulin-binding alkali-stabilized Protein A domains covalentlycoupled to a porous support, wherein the alkali-stabilized Protein Adomains comprise mutants of a parental Fc-binding domain ofStaphylococcus Protein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO51 or 52 are asparagines and wherein at least the asparagine residue atposition 3 of SEQ ID NO 51 or 52 has been mutated to an amino acidselected from the group consisting of glutamic acid, lysine, tyrosine,threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine,valine, alanine, histidine and arginine; and wherein the storage liquidcomprises at least 50% by volume of an aqueous alkali metal hydroxidesolution.